Altered Immune-Related Gene Expressions Indicate Oral Cancer Nodal Disease.

Lymph nodal disease (LN+) is the most significant prognostic factor of oral squamous cell carcinoma (OSCC). Current risk indicator(s) for guiding elective neck dissection (END) is insufficient for clinically node-negative (cN0) patients, resulting in under- or overtreatment. While the role of immunological events in tumorigenesis and metastasis is evident, the prognostic implication in OSCC remains unclear. The study objective was to investigate large-scale immune-related gene expression and determine its prognostic value on node-free survival (NFS). We analyzed patients who received intent-to-cure surgery with at least 3 y of follow-up and known outcome of LN through a pan-Canadian surgical trial. Total RNA was extracted from surgical tissues with >70% tumor content and analyzed on a 730-gene panel (NanoString nCounter® PanCancer Immune Panel). We first profiled gene expression in a fresh-frozen (FF) discovery set to identify differentially expressed (DE) genes, which were then used in unsupervised clustering analysis to identify patient subgroups. The prognostic value of the identified DE genes was then validated on formalin-fixed, paraffin-embedded (FFPE) samples. A total of 177 RNA samples were derived from 89 FF and 88 FFPE surgical tissues, of which 45 (51%) and 40 (45%), respectively, were from patients who developed LN+. We identified 6 DE genes overexpressed in LN+ tumors (false discovery rate <0.001; log2 fold change >1). Clustering analysis separated the patients into 2 subgroups (CM1, CM2), with CM2 exhibiting significantly increased expression and worse 5-y NFS rate (28%; P < 0.001). The prognostic value of these 6 candidate genes was validated on FFPE samples, which were also separated into 2 distinct prognostic groups, confirming the association between increased gene expression and poor 5-y NFS (CM1, 70.3%; CM2, 43.3%; P = 0.01). This is the first study identifying a panel of immune-related genes associated with NFS that can potentially be used clinically stratifying the risk of LN+ at the time of OSCC diagnosis.

Life After Surviving Fontan Surgery: A Meta-Analysis of the Incidence and Predictors of Late Death.

AIM: We now know that 20-40% of patients with a single ventricle will develop heart failure after the second decade post-Fontan surgery. However, we remain unable to risk-stratify the cohort to identify patients at highest risk of late failure and death. We conducted a systematic review of all reported late outcomes for patients with a Fontan circulation to identify predictors of late death. METHODS: We searched MEDLINE, Embase and PubMed with subject terms ("single ventricle", "Hypoplastic left heart syndrome", "congenital heart defects" or "Fontan procedure") AND ("heart failure", "post-operative complications", "death", "cause of death", "transplantation" or "follow-up studies") for relevant studies between January 1990 and December 2015. Variables identified as significant predictors of late death on multivariate analysis were collated for meta-analysis. Survival data was extrapolated from Kaplan-Meier survival curves to generate a distribution-free summary survival curve. RESULTS: Thirty-four relevant publications were identified, with a total of 7536 patients included in the analysis. Mean follow-up duration was 114 months (range 24-269 months). There were 688 (11%) late deaths. Predominant causes of death were late Fontan failure (34%), sudden death (19%) and perioperative death (16%). Estimated mean survival at 5, 10 and 20 years post Fontan surgery were 95% (95%CI 93-96), 91% (95%CI 89-93) and 82% (95%CI 77-85). Significant predictors of late death include prolonged pleural effusions post Fontan surgery (HR1.18, 95%CI 1.09-1.29, p<0.001), protein losing enteropathy (HR2.19, 95%CI 1.69-2.84, p<0.001), increased ventricular end diastolic volume (HR1.03 per 10ml/BSA increase, 95%CI 1.02-1.05, p<0.001) and having a permanent pacemaker (HR12.63, 95%CI 6.17-25.86, p<0.001). CONCLUSIONS: Over 80% of patients who survive Fontan surgery will be alive at 20 years. Developing late sequelae including protein losing enteropathy, ventricular dysfunction or requiring a pacemaker predict a higher risk of late death.

A hybrid approach for fusing 4D-MRI temporal information with 3D-CT for the study of lung and lung tumor motion.

PURPOSE: Accurate visualization of lung motion is important in many clinical applications, such as radiotherapy of lung cancer. Advancement in imaging modalities [e.g., computed tomography (CT) and MRI] has allowed dynamic imaging of lung and lung tumor motion. However, each imaging modality has its advantages and disadvantages. The study presented in this paper aims at generating synthetic 4D-CT dataset for lung cancer patients by combining both continuous three-dimensional (3D) motion captured by 4D-MRI and the high spatial resolution captured by CT using the authors' proposed approach. METHODS: A novel hybrid approach based on deformable image registration (DIR) and finite element method simulation was developed to fuse a static 3D-CT volume (acquired under breath-hold) and the 3D motion information extracted from 4D-MRI dataset, creating a synthetic 4D-CT dataset. RESULTS: The study focuses on imaging of lung and lung tumor. Comparing the synthetic 4D-CT dataset with the acquired 4D-CT dataset of six lung cancer patients based on 420 landmarks, accurate results (average error <2 mm) were achieved using the authors' proposed approach. Their hybrid approach achieved a 40% error reduction (based on landmarks assessment) over using only DIR techniques. CONCLUSIONS: The synthetic 4D-CT dataset generated has high spatial resolution, has excellent lung details, and is able to show movement of lung and lung tumor over multiple breathing cycles.

A spatiotemporal-based scheme for efficient registration-based segmentation of thoracic 4-D MRI.

Dynamic three-dimensional (3-D) (four-dimensional, 4-D) magnetic resonance (MR) imaging is gaining importance in the study of pulmonary motion for respiratory diseases and pulmonary tumor motion for radiotherapy. To perform quantitative analysis using 4-D MR images, segmentation of anatomical structures such as the lung and pulmonary tumor is required. Manual segmentation of entire thoracic 4-D MRI data that typically contains many 3-D volumes acquired over several breathing cycles is extremely tedious, time consuming, and suffers high user variability. This requires the development of new automated segmentation schemes for 4-D MRI data segmentation. Registration-based segmentation technique that uses automatic registration methods for segmentation has been shown to be an accurate method to segment structures for 4-D data series. However, directly applying registration-based segmentation to segment 4-D MRI series lacks efficiency. Here we propose an automated 4-D registration-based segmentation scheme that is based on spatiotemporal information for the segmentation of thoracic 4-D MR lung images. The proposed scheme saved up to 95% of computation amount while achieving comparable accurate segmentations compared to directly applying registration-based segmentation to 4-D dataset. The scheme facilitates rapid 3-D/4-D visualization of the lung and tumor motion and potentially the tracking of tumor during radiation delivery.

Photodynamic therapy: a review and its prospective role in the management of oral potentially malignant disorders.

With the unreliability of epithelial dysplasia as a predictor to determine the risk of future malignant development, subjectivity associated in evaluating dysplasia by pathologists and paucity of biomarkers that could accurately predict the progression risks in oral potentially malignant disorders (PMDs), eradication of the lesions appears to be the most desirable approach to minimize the risk of invasive cancer formation. Interventions, such as surgery and chemoprevention, have not shown promising long-term results in the treatment of these lesions, and lack of guidelines and general consensus on their management has incited much anxiety and doubts in both patients and community clinicians. Topical photodynamic therapy (PDT) is a minimally invasive and minimally toxic technique that in recent years has shown great promise in the management of PMDs. In this review, we describe the historical developments in the field of PDT, its basic mechanisms, as well as related clinical studies, and its challenges in the management of oral PMDs. Based on its high efficacy and low side effects, its high patient acceptance/compliance, the simplicity of the procedure and its minimal pretreatment preparation, topical PDT is believed to have potential to play an important role in the management of PMDs, especially of the low-grade dysplasia.

Ex vivo confocal imaging with contrast agents for the detection of oral potentially malignant lesions.

OBJECTIVES: We investigated the potential use of real-time confocal microscopy in the non-invasive detection of occult oral potentially malignant lesions. Our objectives were to select the best fluorescence contrast agent for cellular morphology enhancement, to build an atlas of confocal microscopic images of normal human oral mucosa, and to determine the accuracy of confocal microscopy to recognize oral high-grade dysplasia lesions on live human tissue. MATERIALS AND METHODS: Five clinically used fluorescent contrast agents were tested in vitro on cultured human cells and validated ex vivo on human oral mucosa. Images acquired ex vivo from normal and diseased human oral biopsies with bench-top fluorescent confocal microscope were compared to conventional histology. Image analyzer software was used as an adjunct tool to objectively compare high-grade dysplasia versus low-grade dysplasia and normal epithelium. RESULTS: Acriflavine Hydrochloride provided the best cellular contrast by preferentially staining the nuclei of the epithelium. Using topical application of Acriflavine Hydrochloride followed by confocal microscopy, we could define morphological characteristics of each cellular layer of the normal human oral mucosa, building an atlas of histology-like images. Applying this technique to diseased oral tissue specimen, we were also able to accurately diagnose the presence of high-grade dysplasia through the increased cellularity and changes in nuclear morphological features. Objective measurement of cellular density by quantitative image analysis was a strong discriminant to differentiate between high-grade dysplasia and low-grade dysplasia lesions. CONCLUSIONS: Pending clinical investigation, real-time confocal microscopy may become a useful adjunct to detect precancerous lesions that are at high risk of cancer progression, direct biopsy and delineate excision margins.

Can clinical and radiological features predict recurrence in solitary keratocystic odontogenic tumors?

INTRODUCTION: The solitary keratocystic odontogenic tumor (KCOT) is a neoplasm, which recurs in 28% of cases. The purpose of this study is to determine whether clinicoradiographic features can predict recurrence. MATERIAL AND METHODS: From 2000 to 2009, 106 solitary KCOTs were retrieved from the Oral Biology Service of British Columbia. Among 58 KCOTs including all recurrent KCOTs (rKCOT) and nonrecurrent KCOTs (nrKCOT) (followed up for at least 5 years), only 29 had radiographs (rKCOT, 18; nrKCOT, 11). RESULTS: Patients with recurrences were significantly older than those without. Interestingly, those cases, which were considered KCOTs before surgery, were significantly more likely not to recur within 5 years of follow-up. Most radiological features did not differ between nrKCOT and rKCOT on the basis of panoramic radiography only.

Stapled peptides with improved potency and specificity that activate p53.

By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.

MicroRNAs in an oral cancer context - from basic biology to clinical utility.

Oral cancer is one of the most commonly diagnosed malignancies worldwide. Its dismal five-year survival rate of ~50% has barely changed for decades. A better understanding of the molecular basis of tumorigenesis - with particular emphasis on disease initiation and progression - is needed to improve clinical outcomes, since this will facilitate the development of drugs and management strategies based on the specific genetic changes underpinning disease behaviors. MicroRNAs (miRNAs), a class of short non-coding RNAs that down-regulate gene expression, have been demonstrated to play essential roles in human cancers. miRNA deregulation has been observed in many tumor types and is implicated in oncogenic cell processes, including proliferation, survival, apoptosis, metastasis, and chemoresistance. In addition, miRNA alterations have been associated with specific clinical phenotypes such as disease progression or recurrence, development of metastases, and post-operative survival. Recent studies have explored the utility of miRNAs as diagnostic and prognostic tools and as potential therapeutic targets. Herein, we discuss miRNA biology and provide a summary of the key findings on the role of miRNAs in oral malignancies.

Unique FISH patterns associated with cancer progression of oral dysplasia.

Subgroups of patients with oral pre-malignant lesions (OPLs) are at extremely high risk for developing invasive cancer in spite of surgical excision. The objective of this study was to evaluate the utility of specific genes and their associated centromeres as markers to stratify OPLs for their cancer risk. Samples used in this study included 35 oral dysplasia with known outcome and 20 normal oral mucosa. Of the dysplasias, 20 were from an ongoing longitudinal study showing progression. The remaining 15 cases (2 of which progressed) were chosen from the population-based, provincial BC Oral Biopsy Service (OBS). Copy number alterations at EGFR, CEP7, CCND1, and CEP11 were evaluated by fluorescent in situ hybridization (FISH). There was no significant difference in demographics between progressors and non-progressors. Specific FISH profiles at these genes and their corresponding centromeres were associated with progression. High gene gain of CCND1 was associated with an 8-fold elevated risk of progression compared with those with no gain in time-to-progression analysis. Numerical alterations of EGFR and CCND1 and their centromeres might be an effective means for identifying OPLs at risk. Future studies will expand on this analysis and set the stage for application of this approach in routine clinical practice.

Dentigerous cyst: a retrospective clinicopathological analysis of 2082 dentigerous cysts in British Columbia, Canada.

The aim of this research is to analyze the prevalence of dentigerous cysts (DCs) in a population-based cohort in British Columbia, Canada, and to report unusual cases associated with DC. The database of the British Columbia Oral Biopsy Service was searched from 1998 to 2007. 2082 histologically confirmed DCs from 2029 patients were retrieved and retrospectively analyzed for incidence, age, gender and ethnicity. The results show that this is a common jaw cyst with male predilection, has a peak incidence in younger adults and is more common in Caucasians. Multiple DCs, representing 2.5% of the cases, are not associated with any syndromes or systemic conditions. 0.5% DCs were associated with other cysts or tumours at the same site or the opposite side of the jaw. The authors report the first series of cases presenting clinically as bilateral DCs, but histologically as an odontogenic tumour or another type of odontogenic cyst. DCs can co-exist with other more serious conditions, such as odontogenic keratocyst or cystic ameloblastoma. This association with more significant conditions indicates the importance of histologically confirming any jaw cyst, even when it presents clinically as a classic DC.

Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni.

In helminth parasites, proteolytic enzymes have been implicated in facilitating host invasion, moulting, feeding, and evasion of the host immune response. These key functions render them potential targets for anti-parasite chemotherapy and immunotherapy. Schistosomes feed on host blood and the digested haemoglobin is their major source of amino acids. Haemoglobin digestion is essential for parasite development, growth, and reproduction. We recently reported the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of Schistosoma mansoni. Here, we report the design of a viral vector expressing a dsRNA hairpin to silence expression of the schistosome cathepsin B1 (SmCB1) gene. We observed 80% reduction in transcript level 72 h after virus exposure and complete silencing of enzyme activity in transduced worms. This is the first report using this technology in any helminth parasite. It will facilitate the evaluation of potential drug targets and biochemical pathways for novel interventions in schistosomes.

Gastrocolic fistula: a rare complication of gastric carcinoma.

Malignant gastrocolic fistula formation is a rare complication of gastric carcinoma. We report a cachectic 82-year-old woman who presented with upper abdominal pain, diarrhoea, loss of weight and loss of appetite. Further investigation of her symptoms revealed a gastrocolic fistula connecting the ulcerated tumour of the body of the stomach to the splenic flexure of the colon.

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines.

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.

Na+/H+ exchanger regulatory factor-1 is a hematopoietic ligand for a subset of the CD34 family of stem cell surface proteins.

CD34 and its relatives, podocalyxin and endoglycan, comprise a family of surface sialomucins expressed by hematopoietic stem/progenitor cells and vascular endothelia. Recent data suggest that they serve as either pro- or antiadhesion molecules depending on their cellular context and their post-translational modifications. In addition, their ability to function as blockers of adhesion may be further regulated by their subcellular localization in membrane microdomains via activation-dependent linkage with the actin cytoskeleton. To gain further insights into the function and regulation of CD34-type molecules, we sought to identify the intracellular ligands that govern their localization. Using both genetic and biochemical approaches, we have identified the Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1) as a selective ligand for podocalyxin and endoglycan but not for the closely related CD34. Furthermore, we show that NHERF-1 is expressed by all c-kit(+) /lineage marker(-)/Sca-1(+) cells, which are known to express podocalyxin and have long-term repopulating abilities. Finally, we show that these proteins relocalize and colocalize in response to cytokine signaling. The results suggest that this cytosolic adaptor protein may be important for mobilization of CD34-type proteins in the plasma membrane and may thereby regulate their ability to block or enhance hematopoietic cell adhesion.

Acute myelogenous leukemia complicated by acute necrotizing ulcerative gingivitis due to Aspergillus terreus.

Infections caused by Aspergillus terreus are rare but have been associated with a poor outcome in immunocompromised patients due to frequent resistance to conventional antifungal therapy. This report describes a case of a woman who developed acute necrotizing ulcerative gingivitis (ANUG) due to A. terreus during induction chemotherapy for acute myelogenous leukemia. She initially failed to respond to treatment with amphotericin B but the infection resolved following the introduction of oral itraconazole. Opportunistic infections caused by A. terreus are an emerging problem and can be associated with a high mortality rate. Early microbiological diagnosis is critical since resistance to amphotericin B is likely and itraconazole appears to be an effective treatment for this infection.

Are metastable, precrystallisation, density-fluctuations a universal phenomena?

In-situ observations of crystallisation in minerals and organic polymers have been made by simultaneous, time-resolved small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS) techniques. In isotactic polypropylene slow quiescent crystallisation shows the onset of large scale ordering prior to crystal growth. Rapid crystallisations studied by melt extrusion indicate the development of well resolved oriented SAXS patterns associated with long range order before the development of crystalline peaks in the WAXS region. Block copolymers self-assemble into mesophases in polymer melts above a critical chain length (or above a critical temperature) and this self-assembly process is shown to be susceptible to an incipient crystallisation. Mesophase formation is observed at anomalously high temperatures in ethylene-oxide containing block copolymers below the normal melting point of the polyoxy ethylene chains. Formation of calcium carbonate from aqueous solutions of sodium carbonate and calcium nitrate is observed to be a two-stage process and precipitation proceeds by the production of an amorphous metastable phase. This phase grows until it is volume filling and leads to the formation of the two polymorphs Calcite and Vaterite. These three sets of results suggest pre-nucleation density fluctuations, leading to a metastable phase, play an integral role in all three classes of crystallisation. In due course, this phase undergoes transformation to "normal" crystals.

Comparison of HPV infection, p53 mutation and allelic losses in post-transplant and non-posttransplant oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (SCC) is increasingly found in transplant recipients, although little is known of the natural history of the disease or the mechanism underlying this increase. METHODS: In this article we describe the history of development of 5 oral post-transplant SCCs (PSCCs) and compare their genetic profiles to 34 non-posttransplant SCCs (NPSCCs). RESULTS: Of the five patients with PSCCs, 3 had bone marrow transplants and two, kidney. All three PSCCs from bone marrow recipients were preceded locally by graft-vs.-host disease (GVHD). Two of the GVHD were biopsied and demonstrated dysplasia. Similar frequencies of loss of heterozygosity (LOH) occurred in PSCCs and NPSCCs at 3p, 9p, 17p and 8p, with lower frequencies in PSCCs at 4q (39% vs. 0%), 11q (53% vs. 20%) and 13q (45% vs. 20%), although the latter were not significantly different. Only 1 PSCC had a p53 mutation, compared to historical values of 40-60% for NPSCC. Interestingly, human papillomavirus (HPV) DNA was detected in 3 (60%) PSCCs, in comparison to only 4 (12%) of the 34 NPSCCs (P = 0.0346). CONCLUSIONS: Dysplasia in oral GVHD may be a strong indicator of cancer risk and should not be regarded as reactive changes to lichenoid mucosites. The low level of p53 mutation and increased HPV infection support the involvement of HPV in the development of PSCC, while the similarity in LOH patterns suggests that other aspects of carcinogenesis may be comparable in these two types of SCCs.