CXCL12 in the metastasising OMMs implies that the CXCR4/CXCL12 axis may be involved in OMM metastasis. Increased expression of APOBEC3A in the metastasising OMMs may indicate APOBEC3A-induced double-strand DNA breaks and pro-metastatic hypermutation. DNA double strand breakage triggers the DNA damage response network and two Fanconi anaemia DNA repair pathway members showed elevated expression in the metastasising OMMs. Cross-validation was employed to test a Linear Discriminant Analysis classifier based upon the RT-qPCR-measured expression levels of CXCL12, APOBEC3A and RPL29. Classification accuracies of 94% (metastasising OMMs) and 86% (non-metastasising OMMs) were estimated.Oral malignant melanoma (OMM) is the most common canine melanocytic neoplasm. Overlap between the somatic mutation profiles of canine OMM and human mucosal melanomas suggest a shared UV-independent molecular aetiology. In common with human mucosal melanomas, most canine OMM metastasise. There is no reliable means of predicting canine OMM metastasis, and systemic therapies for metastatic disease are largely palliative. Herein, we employed exon microarrays for comparative expression profiling of FFPE biopsies of 18 primary canine OMM that metastasised and 10 primary OMM that did not metastasise. Genes displaying metastasis-associated expression may be targets for anti-metastasis treatments, and biomarkers of OMM metastasis. Reduced expression of  A predilection for OMM has been consistently suggested for Poodles3, and variously suggested for Golden Retrievers2, German Shepherd Dogs4 and Boxers4. OMM arise most frequently in the gingiva, but also develop in the buccal and labial mucosa, tongue and hard palate5. OMM is characterised by local invasion, recurrence after surgical resection, high metastatic propensity, and rapid progression from localised to advanced-stage disease6. Estimates of OMM metastasis range from 58–74% to regional lymph nodes, 14–67% to the lungs, and 65% to the tonsils7. Dogs with regional metastases treated with surgery and or radiotherapy have shorter survival times than dogs without metastases8. Adjunctive chemotherapy has not been shown to increase survival9, and data on the impact of the xenogeneic DNA vaccine12 for treatment of advanced OMM is equivocal. A median overall survival time of 335 days has been recently reported for OMM patients in receipt of a systemic adjuvant therapy following surgical excision of the primary tumour13.Oral malignant melanomas (OMMs) are neoplastic proliferations of melanocytes, and are the most common oral tumour in dogs15. Canine OMM metastasis cannot be accurately predicted, nor is there an effective approach for early detection of metastasis. Cytologic or histologic examination of the mandibular and retropharyngeal lymph nodes is commonly performed during staging, and other lymph nodes may be selected for examination on the basis of lymphangiography. However, where OMM metastasis to regional lymph nodes occurs, it is not always detected by examination of the mandibular and retropharyngeal lymph nodes17.Clinicopathological indicators of human cutaneous melanoma malignancy  have limited prognostic utility for canine melanoma, although mitotic index and Ki67 expression level appear to be of some value18, oral mucosal melanoma  accounts for only 1–8% of all human melanomas and around 0.5% of all human oral neoplasms21. However, human OMMs are also aggressive rapidly growing, invasive tumours that display metastatic rates of 66% , 53% (lung23), 36% (bone23), and 20% (liver and brain23), respectively. The 5-year survival rate is 15–25%24. Advanced human mucosal melanoma also has a low rate of response to adjuvant chemotherapy25. All human ethnic groups are affected by oral mucosal melanoma, although the Japanese appear to have an elevated susceptibility27.Whilst OMM represents the most common type of melanoma in dogs29 and in 12% of canine OMM30, and mutations in NRAS have been reported in 3.9% of canine OMMs3 and in 10–22% of human mucosal melanomas33. Around 50% of human cutaneous melanomas have an activating BRAF mutation34, but BRAF mutations occur in only 4–9.5% of human mucosal melanomas36, and have not been found in canine OMMs3.Similarities between the somatic mutation profiles of human mucosal melanomas and canine OMMs suggests a possible overlapping molecular aetiology for UV-independent tumourigenesis. Putative activating mutations in Kit have been described in 7–16% of human mucosal melanomas37 comprises a series of steps, which are believed to be at least partially mediated by the acquisition of metastasis-associated genetic and/or epigenetic alterations additional to those that drive tumour development38. These somatic changes may affect gene expression, and metastasis-associated gene expression signatures have been identified for many human tumours41. Transcriptional profiling has defined the stages in human cutaneous melanoma development and progression as a series of distinct ‘molecular events’, and implicated the involvement of sets of genes in the transition from primary to metastatic melanoma44. Gene expression signatures characteristic of human cutaneous melanoma metastases are detectable in primary cutaneous melanomas. A 1,864 gene expression signature derived from profiling cutaneous melanoma metastases was subsequently shown to delineate primary melanomas into two classes associated with significantly different relapse-free and overall survival45. Integration of functional and structural protein interaction data with primary and metastatic melanoma gene expression data enabled derivation of a panel of 6 genes which distinguished human primary and metastatic cutaneous melanoma and predicted melanoma-specific survival46. For human oral mucosal melanoma, comparative gene expression analysis of lymph node metastases and paired non-metastatic lymph nodes has elucidated the involvement of long non-coding RNAs in the regulation of metastasis-associated gene expression47. Metastasis-associated gene expression signatures identified in primary tumours may predict metastasis, and indeed assay of the expression of 15 genes is the basis of a routine test for human uveal melanoma metastasis48.The metastatic cascade49, but no equivalent data is available for the more common canine OMM. Although we also recently identified pro-metastatic gene expression in an unrelated canine cancer (mast cell tumours50), the overall low degree of ‘overlap’ between the metastatic gene expression signatures identified for different human solid tumours51 affords the rationale for the gene expression profiling-based study of metastasis in different canine cancers, mirroring the approach adopted in the study of human tumour metastasis.Gene expression profiling of canine cutaneous melanoma and melanocytoma has implicated the increased expression of genes involved in extracellular matrix-receptor interaction and the phosphoinositide 3-kinase/protein kinase B pathway in metastatic progressionThe high rate of mortality attributable to  canine OMM metastasis is a significant welfare issue. It would be hugely beneficial for clinicians and owners alike to know whether a tumour was going to metastasise, while prevention of OMM metastasis would save the lives of most canine OMM patients. Establishing the role of dysregulated gene expression in canine OMM metastasis is an opportunity for identifying metastasis-associated biomarkers and possible anti-metastasis therapeutic targets.In this project we performed comparative genome-wide expression profiling of archival biopsies of canine primary OMMs that metastasised and did not metastasise. We sought to identify metastasis-associated gene expression, and assess whether metastasising and non-metastasising OMMs could be delineated by the expression levels of genes associated with metastatic progression.52 analysis of human tumour gene expression datasets (from the Gene Expression Omnibus53), it was estimated that a ‘Discovery Rate’ of 73.1–81.7% at the 0.05 significance level would be afforded using 20 tumour samples in each of two ‘outcome groups’.OMM biopsies from 42 dogs [29 bearing a metastasising (M) tumour and 13 bearing a non-metastasising (NM) tumour] qualified for the study. Through PowerAtlasSeveral requests for FFPE OMM biopsies from patients fulfilling the NM inclusion criteria were made to all UK veterinary university teaching hospitals and three large multidisciplinary private practices in the UK, but no additional NM OMM biopsies could be recruited because of the regular use of xenogeneic vaccination for patients regardless of metastatic status at the time of presentation. One NM OMM biopsy was subsequently excluded because of a sub-optimal RNA concentration. The integrity of each OMM RNA sample was determined and 20 M OMM and 12 NM OMM sample groups, with similar RNA integrity ranges, assembled Tables  and S2.54, and 2 M and 2 NM OMMs excluded , whilst 61% and 60% of the M and NM OMM dogs were male or neutered male, respectively. No association between OMM gene expression profile and either gender, or age at diagnosis, could be gleaned by hierarchical clustering of the 28 OMMs according to the expression values of the 20% of Transcript Clusters  that had the highest variance in expression signal. Biopsies from 5 of the 8 Golden Retrievers were grouped in a single cluster (together with biopsies from a Dachshund and Bullmastiff), whilst the 3 remaining Golden Retriever OMM biopsies were partitioned in a second large sub-cluster with biopsies borne by 6 other breeds  between the M and NM OMMs were compared to those assigned to the Transcript clusters  for which the expression (above background) of ≥1 exon probe set was detected in at least 30% of the OMMs in the M and/or M groups. Over-represented amongst the genes exhibiting differential expression were 4 Gene Ontology Consortium biological processes and one KEGG pathway  of >35 meant that valid gene expression measurements were recorded for slightly fewer than the 28 OMM biopsies profiled by microarray hybridisation. For 8 genes, the expression levels in individual OMMs measured by microarray (exon-level probe set) and RT-qPCR, respectively, were highly concordant, as were the NM OMM/M OMM fold differences in expression determined by the two techniques Table . For SLC56, including fold-change differences in gene expression between classes58. Consequently, the efficacy of using the 3 genes shown  to exhibit >two-fold differential expression  was predicted to be Linear Discriminant Analysis . The ranking of genes for their utility in class prediction may be based on the statistical significance of their difference in expression between classes. However, ‘filter’ gene selection methods may be based upon other metricsRandom sampling cross-validation was initially employed to test the performance of the LDA classifier. Two M OMMs and 1 NM OMM were randomly selected on each of 20 occasions, and the accuracy of their classification (as N or NM) measured after training the classifier using the remaining 15 M and 6 NM OMMs’ expression values Fig. . For M O18 and the most common oral malignancy in dogs1. At the present time there is no means to accurately predict if an individual OMM is one of the significant proportion of tumours that will metastasise, and metastasis is commonly underestimated during tumour staging due to a failure to sample all appropriate lymph nodes, and/or a failure of detection by standard cytology or histopathology. Metastasis is the most common cause of death in dogs treated for OMM, but there is currently no proven effective therapy to delay or prevent metastasis. With no means of accurately predicting OMM metastasis, it is possible that palliative adjuvant treatment is not prescribed for dogs with an unrecognised ‘metastasising OMM’.Malignant melanomas are the most common canine melanocytic neoplasmMolecular genetic and epigenetic ‘events’ that promote canine OMM metastasis may be both predictive indicators of canine OMM metastasis and the focus for therapeutics intended to prevent metastasis. Through comparative genome-wide gene expression profiling of 18 primary OMMs that metastasised and 10 primary OMMs that did not metastasise, in the present study we aimed to identify dog breed-independent OMM metastasis-associated gene expression.47. An understanding of the mechanisms by which changes in gene expression mediate OMM metastasis is afforded by functional annotation enrichment analysis.Increased expression in M OMMs characterised 60% of the genes differentially expressed between M and NM OMMs. Further indication of the potential significance to OMM metastasis of the ‘up-regulation’ of pro-metastatic gene expression is that a similar proportion of the genes differentially expressed between human oral mucosal melanoma lymph node metastases and paired non-metastatic lymph nodes were expressed at an increased level in the lymph node metastases59. Aberrant expression of protein phosphatase inhibitors has been reported in a wide variety of human cancers60. Two genes  with the ‘negative regulation of phosphatase activity’ annotation showed decreased expression in the M OMMs and three genes  with the same annotation showed increased expression in the M OMMs. PPP1R37, GPATCH2 , CASC5 (or PPP1R55) and CSRNP2 (or PPP1R72) encode proteins recognised as inhibitory regulatory subunits of phosphoprotein phosphatase 1 (PPP1)61, a protein serine-threonine phosphatase that regulates several members of the Transforming growth factor beta signalling pathway62, which promotes invasion and metastasis in advanced stages of cancer63. Inhibition of PPP1 by the regulatory subunit PPP1R1A in Ewing sarcoma has been shown to promote tumour growth and metastasis64. CHP1  inhibits the serine/threonine protein phosphatase 2B65, which is responsible for the dephosphorylation (and thus activation) of Nuclear factor of activated T cells (NFAT) transcription factors 1–466. NFATs have been shown to have both pro-metastatic67 and anti-metastastic68 activities.Reversible protein phosphorylation is integral to intracellular signal transduction pathways, and requires the coordinated action of protein kinases and protein phosphatases. Disruption of the balance between phosphorylation and dephosphorylation is associated with carcinogenesis, and protein phosphatases have been recognised as tumour suppressors69. The pathway forms part of the DNA damage response network70 which maintains genome integrity. Four genes  with the ‘FA pathway’ annotation showed elevated expression in M OMMs, whilst a fifth FA pathway gene (FANCC) displayed marginally decreased expression in the M OMMs. FANCB and FANCC encode proteins that form part of the FA core complex, an ubiquitin E3 ligase that monoubiquinates FANCI (and FANCD2), the ID complex, which is subsequently re-localised to the DNA lesion, and (with Group III FA proteins) co-ordinates DNA cross-link repair71. The RPA2 and TOP3A proteins perform DNA repair functions associated with the FA pathway. RPA2 is a subunit of the Replication Protein A complex, which is involved in DNA repair in the cellular response to DNA damage72, whilst TOP3A encodes a DNA topoisomerase that controls DNA topology during DNA repair73. The elevated expression of DNA repair pathway genes in human primary cutaneous melanoma has been associated with distant metastasis and poor prognosis74. Subsequently, the increased expression of FA and DNA damage response pathway genes has been reported in human ‘high grade’ primary cutaneous melanoma , relative to ‘low grade’ primary tumours45, and in human cutaneous melanoma metastases relative to normal skin75. As FA pathway gene expression correlates with FA pathway activity76, it appears that the FA pathway is activated in metastasising human cutaneous melanoma and canine OMM. FA pathway activation may be a response to increased genome instability in advanced melanomas, and may confer a selective advantage supporting metastasis to distant sites.The Fanconi anaemia (FA) pathway is a multi-protein DNA repair pathway that resolves DNA interstrand cross-links encountered during DNA replication that would otherwise block replication and transcription, and lead to gross chromosome abnormalitiesCOX10, MUL1)) with the ‘mitochondrial fission’ annotation displayed decreased expression in the M OMMs and one gene (MTFR1) with the same annotation showed elevated expression in the M OMMs. Increased expression of mitochondrial fission pathway genes has been associated with the invasiveness and metastasis of some cancers78. MTFR1  is upregulated in metastatic uveal melanoma79, and is a member of a 20-gene panel whose collective high expression is predictive of prostate cancer metastasis80. Conversely, suppression of MUL1  has been associated with the progression of human head and neck cancer81. COX10 (Cytochrome C Oxidase Assembly Homolog 10) is a member of a 14-gene classifier for colorectal cancer metastasis, identified by differential gene expression analysis of early and late stage primary colorectal cancer82.Two genes  with the ‘Positive regulation of cholesterol efflux’ and/or ‘(positive regulation of) cholesterol homeostasis’ annotation were expressed at a decreased level in the M OMMs and three genes  with one, or both, of the same annotations, showed elevated expression in the M OMMs. NR1H3 (or Liver X Receptor Alpha isoform) is a Nuclear Receptor superfamily transcription factor which when activated by oxysterol binding drives cholesterol efflux85. Agonist activation of the Liver X Receptor Beta isoform (NR1H2) has been shown to suppress the growth and metastasis of melanoma cells by transcriptional induction of apolipoprotein-E86. Reduced NR1H3 expression is predictive of decreased recurrence-free survival in muscle-invasive bladder cancer87, and associated with reduced overall survival in hepatocellular carcinoma88. APOE (apolipoprotein-E) is a lipid transport protein essential for the normal catabolism of triglyceride-rich lipoproteins89. Elevated APOE expression is associated with lymph node metastasis of human gastric cancer90 and lung adenocarcinoma91, although it has been identified as a metastasis suppressor in vitro in human cutaneous melanoma92. PLTP (Phospholipid Transfer Protein) transfers phospholipids from triglyceride-rich lipoproteins to high density lipoprotein, and is involved in the uptake of cholesterol from peripheral cells and tissues. PLTP expression was increased in Grade IV human glioma relative to low grade glioma, and knockdown in vitro lead to the decreased migration of glioblastoma tumour cells93. In concept, the increased expression of PLTP in the NM OMMs observed in the present study may be consistent with the production by the tumours of interleukin 6, which has been shown to inhibit melanoma growth94. The increased expression of PLTP in the spontaneous regression phase of canine transmissible venereal tumour has previously been associated with increased IL-6 production95. The ATP-binding cassette transporter 2 (ABCA2) is a membrane-associated protein involved in sphingolipid transport. ABCA2 deficiency inhibits prostate tumour metastasis in vivo, potentially through reduction of the intracellular sphingolipid level96, whilst ABCA2 expression is increased in ovarian carcinoma metastases relative to primary tumours97.Deregulation of the expression of genes involved in cholesterol homeostasis pathways has been associated with cancer development and progressionCXCL12, APOBEC3A and RPL29). As fold change has been effectively employed to rank genes for their potential efficacy in gene expression level-based classification58, the 3 genes were selected for use in class prediction. Cross-validation was deployed in a preliminary evaluation to test the accuracy of a Linear Discriminant Analysis-based classifier featuring the 3 genes. The classification accuracies estimated were 94–100% (M OMMs) and 86–100% (NM OMMs), respectively. The LDA classifier performance will need to be validated by further retrospective, and prospective study. If the classification accuracy is confirmed, the cross-breed OMM metastasis-associated 3-gene expression signature would form the basis of an objective and quantitative predictive test for OMM metastasis that could make a significant contribution to the clinical management of canine OMM.In the present study, RT-qPCR analysis confirmed >two-fold differential expression between M and NM OMMs for 3 genes  is secreted by stromal cells and is a ligand for the G-protein coupled receptors CXCR4 and CXCR798. Binding of CXCL12 to CXCR4 activates four signal transduction pathways that induce cytoskeletal rearrangement, cell growth, angiogenesis, and anti-apoptotic effects98. Interaction between CXCL12 and CXCR4 has also been shown to mediate metastasis, and direct metastatic dissemination to organs expressing high levels of CXCL1299. Blocking CXCL12 binding to CXCR4 reduced the migration of human uveal melanoma cells in vitro100, and pulmonary metastasis of murine B16 cutaneous melanoma cells101. Low CXCL12 expression in human primary cutaneous melanomas has been associated with poor prognosis102 and shown to be predictive of metastasis103, and CXCL12 is one of 789 genes displaying reduced expression in ‘high grade’ human primary cutaneous melanomas that is a member of a 1,864 gene expression signature that delineates two classes of primary cutaneous melanomas with significantly different rates of metastasis104.APOBEC3A  encodes a cytidine deaminase which preferentially binds to the sequence 5′-(C/T)TCA in RNA or single-stranded DNA and converts cytosine to uracil. Deregulated APOBEC3A expression in cancer is believed to induce double strand breaks in genomic DNA activating DNA damage response pathways105. The repair of such breaks triggers the formation of single stranded DNAs which are substrates for APOBEC3A-mediated hypermutation, such that 5′-(C/T)TTA APOBEC3A mutation signatures occur in clusters (on one DNA strand) in multiple human cancers106. The extent of APOBEC-associated mutations correlates with APOBEC mRNA expression levels107. APOBEC3A-mediated mutagenesis occurs at different stages in different cancers108, and is thought to drive tumour evolution, including promoting metastasis109.RPL29  encodes a component of the 60 S ribosomal subunit. Beyond their role in ribosome assembly and protein translation, differential expression of ribosomal protein genes in cancer has been associated with ribosome-independent regulation of cell growth and proliferation, apoptosis, invasion and metastasis110. Ribosome-free ribosomal proteins have been implicated as being both oncogenic and tumour-suppressors110. Silencing of RPL29 suppressed the proliferation of human pancreatic tumour cells and enhanced apoptosis111 suggesting an involvement in cell proliferation. However, RPL29 silencing had no effect on the viability of human metastatic melanoma cells112. The expression of a specific ribosomal protein has been shown to be a prognostic indicator for multiple human cancers113.114. Cellular receptors in signal transduction pathways that control cell to cell and cell to ECM adhesion are targets for anti-metastatics in development115. Pro-metastatic gene expression in OMMs is a potential target for anti-metastasis therapeutics. Targeting the interaction between the CXCR4 receptor and CXCL12 has been evaluated as a strategy for inhibiting CXCR4-CXCL12 axis-mediated melanoma metastasis. Small molecule inhibitors of CXCR4 were shown to be effective at disrupting the liver metastasis of uveal melanoma cells in mice116, and migration of human cutaneous melanoma cells in vitro117. Chemical inhibition of the mutational activity of APOBEC3A (and APOBEC3B) is being evaluated as a cancer therapeutic118. Furthermore, demonstration of microRNA post-transcriptional regulation of APOBEC gene expression119 suggests the use of miR-mimics120 as a potential means of APOBEC deaminase inactivation119. Intriguingly, it has been postulated that APOBEC3A-mediated hypermutation could generate new tumour-specific antigens thereby enhancing the efficacy of immune stimulation therapies106. The potential for suppressing melanoma metastasis through activation of Liver X Receptors (LXR) using synthetic agonists has been investigated. Activation of the LXR Beta isoform (NR1H2) was shown to inhibit human and murine cutaneous melanoma cell migration in vitro and murine cutaneous melanoma cell metastasis in a mouse xenograft model121. If NR1H3 is the predominant LXR Receptor isoform expressed it remains to be seen if LXR agonists would initiate receptor activation with anti-metastatic effect. The up-regulation of FA pathway DNA repair genes in human cutaneous melanomas and canine OMM may be both pro-metastatic by negating the impact of increased genome instability, and contribute to melanoma metastases chemoresistance. Consequently, in concept, the use of FA pathway inhibitors122 may be an option for treatment of melanoma.In human medicine, anti-angiogenics and matrix metalloproteinase inhibitors have been licenced for treatment of tumour metastasis124. Validation of the predictive accuracy of the 3-gene LDA classifier will be achieved through further retrospective, and prospective, studies featuring larger numbers of  M and NM OMM biopsies. Ultimately, experimental investigations involving canine OMM cells will be necessary to confirm the functional consequences (e.g. in regard to cell migration) of metastasis-associated differential gene expression that are anticipated given gene function(s) and (in some cases) prior in vitro and in vivo study evidence.The major limitation of this study was that the number of NM OMMs included was restricted by the need to attempt to ensure that a primary tumour classified as ‘non-metastasising’ was only classified as such because it did not exhibit pro-metastatic gene expression as opposed to its metastasis potentially being prevented by a systemic adjuvant therapy. The consequence of the widespread use of the xenogenic melanoma vaccine in the UK was the exclusion of OMM biopsies from many dogs that had not developed metastatic disease. Furthermore, due to difficulties associated with collecting fresh canine tumour biopsies, FFPE biopsies of primary canine OMM  were used in the study. However, data that is both biologically authentic and clinically-relevant has been obtained by Affymetrix microarray-based gene expression profiling of FFPE tissuesin vitro and in vivo model studies, and may be as likely as human melanomas to be responsive to therapeutics whose efficacy for treatment of human melanoma has yet to be investigated. Quantification of the expression of 3 genes, each of which displays a greater than two-fold differential expression between canine OMMs that do and do not metastasise, may be the potential basis for a test that would accurately predict canine OMM metastasis, and thereby assist a clinician to make an informed decision about the most appropriate treatment for a canine OMM patient.Several of the differences in gene expression observed between primary canine OMMs that metastasised and OMMs that did not metastasise in this study have previously been associated with human cutaneous melanoma metastasis. The genes involved have been targets in proof of principle trials of potential anti-metastatic melanoma therapeutics. Other genes that exhibit differential expression between metastasising and non-metastasising primary canine OMMs may represent potential new targets for both canine and human cancer drugs. The results obtained in the present study suggest that OMMs in dogs may be responsive to anti-human melanoma metastasis therapeutics currently in clinical trials, or being evaluated through preclinical This study was conducted with the approval of the ethics committees of the Animal Health Trust and the University of Liverpool, respectively. All methods were performed in accordance with the relevant guidelines and regulations. An OMM biopsy was only included in the study with the informed, written consent of the owner of the dog who bore the tumour. The treatment that a OMM patient received was unaffected by the inclusion of a biopsy of their tumour in the study.and diagnostic imaging-confirmed metastasis, and who died or were euthanased because of OMM metastasis <500 days after surgery/biopsy . ‘Non-metastasising’ (NM) OMM biopsies were from dogs without pathological analysis and imaging-confirmed metastasis, whom did not receive any adjuvant therapy, and who were still alive >540 days post-surgery/biopsy.FFPE biopsies of OMMs were collected (between 1993 and 2010) for histopathology from dogs attending the Clinical Oncology departments at the Animal Health Trust Centre for Small Animal Studies, University of Liverpool Small Animal Teaching Hospital, Dick White Referrals, and Colorado State University Veterinary Teaching Hospital. Biopsies were recovered by surgery performed (prior to any adjuvant treatment) on dogs for whom complete staging and follow-up information were available. Metastasis was confirmed by abdominal ultrasound or computed tomography, and cytological/histological examination of ≥1 regional lymph nodes. ‘Metastasising’ (M) OMM biopsies were from dogs that had pathological analysis The RecoverAll Total Nucleic Acid Isolation Kit, which facilitates on-column DNase digestion , was used to isolate total RNA from FFPE OMM biopsies. RNA samples were further purified by spin column filtration  and additional DNase treatment . Finally, RNAs were concentrated  and quantified by RiboGreen fluorometry .125 was employed to assess the integrity of each FFPE RNA sample as described previously50. Procedural details are summarised in the Supplementary Information.Reverse transcription-quantitative PCR (RT-qPCR) measurement of a 126 bp fragment of a 130–150 bp short interspersed nuclear element (SINE) that occurs every 5–8.3 kb in the canine genomein vitro transcription. Each cDNA was individually hybridised to an array in a Canine Gene 1.1 ST Array Strip , in a proprietary hybridisation cocktail , for 20 h at 48 °C. Array strip washing and streptavidin-phycoerythrin staining were undertaken by the GeneAtlas System  Fluidics Station, and array scanning by the GeneAtlas System  Imaging Station.Fragmented, biotinylated single-stranded cDNA was prepared from 5.1 ng of each FFPE OMM RNA sample using the GeneChip WT Pico Reagent Kit . cDNA preparation involved target amplification by 11 cycles of adaptor-primer PCR and 14 h of 126) by quantile normalisation, log2 transformation and signal summarisation, respectively. Arrays with ≥1 sample quality, labelling quality or hybridisation quality metric value ≥2 standard deviations from the mean for all the arrays54 were excluded, and the raw probe-level signal intensity data for the remaining arrays re-processed to generate quantile normalised and log2-transformed exon and gene-level probe set expression values. Gene-level probe sets (‘Transcript clusters’) annotated as ‘crosshyb_type’ = 1 (unique hybridisation target) and ‘category’ = ‘main’127, and for which the expression above background  of ≥l exon probe set could be detected in at least 30% of the OMMs in the M and/or NM group, were judged to be expressed in the OMMs and were included in further analyses.Microarray data was processed using the ‘Affymetrix Expression Console Software 1.3’ . ‘Outlier arrays’ were first identified by review of exon-level probe set expression values created  was employed to attempt to establish the potential identity of Transcript clusters representing ‘predicted genes’, or for which gene annotation was unavailable.Hierarchical clustering  was performed using Cluster133. The functional annotations associated with differentially expressed genes were compared with those ascribed to all Transcript clusters (‘crosshyb_type’ = 1 and ‘category’ = ‘main’) for which the expression of ≥1 exon probe set was detected above background in ≥30% of the tumours in the NM and/or M OMM cohort, and over-represented biological processes and pathways identified.The biological processes and pathways affected by the differences in gene expression observed between M and NM OMMs were identified using DAVID125, present in the 3′-untranslated region of hundreds of canine mRNAs, was also assayed as a ‘reference gene’ for target gene expression measure normalisation134. Geometric mean Cq ≥35 were considered an unreliable measurement of gene expression and were excluded, as were the results of triplicate PCR assays with a Cq standard deviation of >0.5. Additional information is provided in the Supplementary Information.RT-qPCR was employed for validation of differential gene expression. A unique region within the exon probe set that displayed the largest statistically significant fold-difference in expression between M and NM OMMs was the template for design  of a TaqMan or SYBR Green PCR assay. The expression level of each gene in each OMM sample was measured as the geometric Cq value calculated from triplicate PCR reactions performed using preamplified cDNAs, prepared from cDNAs previously screened for PCR inhibitors. A 71 bp fragment of a SINE135. Using qbase + , target gene geometric mean Cq values were converted to relative gene expression measurements  by application of a canine SINE134 geometric mean Cq-derived normalisation factor. A two-tailed t-test for unpaired data was performed on NRQ log10 transformations to identify genes exhibiting statistically significant differences in expression between M and NM OMMs.Relative quantification of gene expression was performed using a modification of the delta-delta-Ct method which accounts for differences between the amplification efficiencies of target and potentially multiple ‘reference’ genes136 predicts the performance in class prediction of representatives of 10 classification functions and, through evaluation of the expression values obtained for the Transcript clusters expressed in the OMM, was employed to identify the optimal classification function for prediction of OMM ‘metastatic status’ (M or NM) on the basis of OMM gene expression. Linear Discriminant Analysis-based class prediction was undertaken using the R Package MASS130lda function implemented in the R environment for statistical analyses137. The accuracy of class prediction, performed using RT-qPCR-derived gene expression measurements, was estimated by cross-validation. In random sampling cross-validation, on each of 20 occasions the class (M or NM) of two M and one NM OMM, which constituted a ‘test data set’, were predicted after the classifier had been trained using the gene expression values (‘training data set’) obtained for the remaining OMMs (90% of the tumours). In leave-one-out cross-validation, the class of each OMM was predicted after the classifier had been trained using the remaining (n-1) OMM gene expression data.The R package SPreFuGEDSupplementary Information


Standard treatment for severe malaria is with artesunate; patient survival in the 24 hours immediately posttreatment is the key objective. Clinical trials use clearance rates of circulating parasites as their clinical outcome, but the pathology of severe malaria is attributed primarily to noncirculating, sequestered, parasites, so there is a disconnect between existing clinical metrics and objectives.We extend existing pharmacokinetic/pharmacodynamic modeling methods to simulate the treatment of 10000 patients with severe malaria and track the pathology caused by sequestered parasites.Our model recovered the clinical outcomes of existing studies (based on circulating parasites) and showed a “simplified” artesunate regimen was noninferior to the existing World Health Organization regimen across the patient population but resulted in worse outcomes in a subgroup of patients with infections clustered in early stages of the parasite life cycle. This same group of patients were extremely vulnerable to resistance emerging in parasite early ring stages.We quantify patient outcomes in a manner appropriate for severe malaria with a flexible framework that allows future researchers to implement their beliefs about underlying pathology. We highlight with some urgency the threat posed to treatment of severe malaria by artemisinin resistance in parasite early ring stages. A new mathematical model of severe malaria calculating pathology of sequestered parasites shows the standard World Health Organization regimen outperforms a simplified artesunate regimen in patients with early stage synchronized infections. Artesunate resistance leads to worse outcomes for these patients. See the Editorial Commentary by Small and Seydel on pages 1176–7.).P. falciparum erythrocyte membrane protein 1 [A key factor responsible for severe malaria is the binding of parasitized erythrocytes  to microvascular endothelium, a process known as sequestration. iRBC sequestration induces pathology through 3 main causes: (1) impairing blood flow to organs through direct physical blockage of the capillaries , (2) indrotein 1 ) are stirotein 1 , 11.Parasite clearance rates are a commonly used clinical outcome measure to compare efficacy of antimalarial treatment regimens. However, parasite clearance rates correlate poorly with disease outcome in severe malaria. Large trials comparing intramuscular artemether with quinine in African children showed more rapid parasite clearance with artemether but no difference in case fatality , 13. WitP. falciparum parasites undergo a 48-hour developmental cycle in human erythrocytes with 2 main implications for pathology and treatment. Firstly, parasites initially circulate freely in blood vessels but sequester  at mature stages of their intraerythrocytic cycle. Secondly, parasites differ in their sensitivity to drugs over the course of this 48-hour cycle.We utilized a computer-based PK/PD model to track changes in the number of sequestered iRBCs following drug administration. The model was implemented in the statistical programming software R  version We assumed that severe malaria pathology is caused by a single clone . We varied the “recovery rate” r in the simulations by altering its half-life (L(t) at any time t posttreatment as the sum of the current number of sequestered iRBCs with living parasites α(t) and the lingering pathological effects of once-sequestered iRBC whose parasites were killed in the current or previous time periods, β(i), that is Severity of the malaria infection is determined by what we refer to as “pathological load,” that is the number of sequestered iRBCs (containing either living or dead parasites) physically restricting blood flow and/or eliciting patient’s immune and/or inflammatory response that may also contribute to pathology , 24. It alf-life , which iL(t) occurring during a defined time period posttreatment, and (2) the area under the pathological load curve (AUCPL) during a defined time period posttreatment, that is the total pathology in that period. For example, the AUCPL in the period 0 to 24 hours posttreatment is: We used 2 metrics to analyze treatment regimens and resistance: (1) maximum pathological load (MPL), the maximum value of in each patient. Follow-up time was 48 hours after drug administration; this reflected a whole parasite life cycle within an iRBC but, more importantly, covers the period posttreatment where a patient is most likely to die [We simulated a cohort of 10000 patients who had parasitological, pharmacological, and patient-specific parameters drawn from the distributions given in y to die , 32.PL and MPL).We conducted partial rank correlation coefficient (PRCC) using Spearman ρ to establish the strength of the relationship between model parameters and dependent variables  reinvading to restart the asexual life cycle. The mean age-bin variable was therefore split into either 5 or 3 ordinal classes (depending on whether parasites were hypersensitive or resistant to artemisinin), as described in The following parameters were included in the PRCC analysis:• duration of artesunate killing posttreatment; this captures all the PK/PD parameters in Vmax)• maximal rate of artesunate killing • variation of initial age-bin distribution (measured as the standard deviation (SD) around the mean)• initial parasite number• parasite multiplication rate (PMR)r).• half-life of the ‘pathological recovery rate’  was >99% [24 >99% for the standard and simplified intramuscular regimens, respectively.Our model calculated parasite reduction ratios (PRR) from circulating parasite numbers . The cliwas >99% , reportewas >99% , our reswas >99%   in our simulation using parameters from Hendriksen et al [Hendriksen et al  do not rer et al  and Hender et al . Howeverer et al  reporteden et al  was >99%48 following artemisinin treatment is 10–4 [48 of 5.18−5 , but PRCC of <0.3 in earlier time periods. All other parameters had PRCC values of <0.3, indicating that outcome metrics were not highly correlated as per accepted statistical criteria [We simulated treatment of drug-sensitive parasites with the standard regimen and identified the key drivers of pathology by calculating which parameters were most correlated with AUC and MPL . The moscriteria . All othPL and MPL. The simplified regimen had a slightly higher median ratio in 0–24 hours of 1.03; MPL was 1. At 24–48 hours, higher medians of 1.49 and 1.45 for AUCPL and MPL, respectively, were observed  when treating artemisinin-resistant parasites. Differences between regimens were extremely similar to those shown in PL and MPL when comparing resistant and sensitive parasites are never less than 1 . This occurs because artesunate presence posttreatment largely coincides with parasites in age-bins insensitive to artesunate through resistance, rendering the initial dose nearly or completely ineffective.In these early age-bins, ratios for AUCSD of the initial mean age-bin had a positive correlation with the ratio (indicating that resistant parasites had worse outcomes as SD increased). This occurred because higher SD “nudged” parts of the age-bin distribution into (or out of) resistant age-bins . PRCC analysis showed no other parameter had a PRCC value of >0.01, suggesting the initial mean age-bin  are the sole determinants of whether a patient’s outcome will be worse in the presence of resistance.PL). An interesting, highly important result is that the key quantitative assumption made in the analysis, the rate of resolution of pathology , had little effect on our conclusions when comparing alternative regimens or the impact of resistance . The first 24 hours are critical for patient survival [Kremsner and colleagues , 37 concreatment . This disurvival , so outcPL. Resistance prevents drug killing in age-bins 2–4 (these bins are otherwise hypersensitive) resulting in no killing for a contiguous 8-hour period in resistant parasites . Our results show the initial mean age-bin and its SD are the only parameters that distinguish outcomes between sensitive and resistance parasites , and, with novel metrics, used to investigate the pathology of severe malaria. Our model showed that while on a population level a simplified artesunate regimen is noninferior to the standard WHO regimen, outcomes in a subgroup of patients with infections grouped in late or early initial mean age-bins are notably worse with the simplified regimen. The emergence of artemisinin resistance in early ring stages poses a significant threat to this same group of patients. Neither of these results are particularly obvious from summary statistics of the population and so subgroup analysis is particularly important in devising treatment strategies for severe malaria.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary MaterialClick here for additional data file.Supplementary Table S1Click here for additional data file.Supplementary Table S2Click here for additional data file.Supplementary Table S3Click here for additional data file.Supplementary Table S4Click here for additional data file.Supplementary Table S5Click here for additional data file.Supplementary Table S6Click here for additional data file.Supplementary Table S7Click here for additional data file.Supplementary Table S8Click here for additional data file.Supplementary Table S9Click here for additional data file.Supplementary Table S10Click here for additional data file.Supplementary Figure S1Click here for additional data file.Supplementary Figure S2Click here for additional data file.Supplementary Figure S3Click here for additional data file.Supplementary Figure S4Click here for additional data file.Supplementary Figure S5Click here for additional data file.Supplementary Figure S6Click here for additional data file.Supplementary Figure S7Click here for additional data file.


PCDHAC2, PCDHGC5 and PCDH10 could be used as survival predictors in PDAC patients.Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and is not a clinically homogeneous disease, but subsets of patients with distinct prognosis and response to therapy can be identified by genome-wide analyses. Mutations in major PDAC driver genes were associated with poor survival. By bioinformatics analysis, we identified protocadherins among the most frequently mutated genes in PDAC suggesting an important role of these genes in the biology of this tumor. Promoter methylation of protocadherins has been suggested as a prognostic marker in different tumors, but in PDAC this epigenetic modification has not been extensively studied. Thus, we evaluated whether promoter methylation of three frequently mutated protocadherins, DNA extracted from 23 PDACs and adjacent non-neoplastic pancreatic tissues were bisulfite treated. Combined Bisulfite Restriction Analysis (COBRA) coupled to denaturing high-performance liquid chromatography (dHPLC) detection and bisulfite genomic sequencing (BGS) were used to determine the presence of methylated CpG dinucleotides in the promoter amplicons analyzed.PCDHAC2, complete methylation for PCDHGC5. Conversely, the third protocadherin analyzed, PCDH10, showed a variable degree of CpG methylation in PDAC and absence of methylation in adjacent non-neoplastic pancreatic tissues. At Kaplan–Meier analysis, high levels of PCDH10 methylation defined according to the receiver operating characteristic (ROC) curve analysis were significantly associated with worse progression-free survival (PFS) rates (P = 0.008), but not with overall survival (OS). High levels of PCDH10 methylation were a prognostic factor influencing PFS , but not the OS.In an exploratory analysis, two protocadherins showed the same pattern of CpG methylation in PDAC and adjacent non-neoplastic pancreatic tissues: lack of methylation for PCDH10 can predict prognosis in PDAC patients with a significant impact on the outcome in terms of progression-free survival. High levels of PCDH10 promoter methylation could be useful to identify patients at high risk of disease progression, contributing to a more accurate stratification of PDAC patients for personalized clinical management.In this study, we show for the first time that the methylation status of  Pancreatic ductal adenocarcinomas (PDAC) arise from the exocrine pancreas, account for 95% of pancreatic cancers and, due to the poor survival rate, represent the seventh leading cause of cancer-related deaths worldwide and the third in the United States . PDAC arSome studies analyzed the mutational landscape of PDAC using state of the art genomic sequencing –6. ConveGenes that are frequently mutated in PDAC are likely to play an essential role in the biology of this tumor, and they might also be a target of epigenetic dysregulation. Therefore, studying epigenetic changes in these genes may provide complementary evidence of their role in PDAC malignant transformation.clustered” and “non clustered” protocadherins [clustered protocadherins constitute the largest group. Unlike the clustered, the non clustered protocaderins are so named because their genes are not located in a single gene locus, but in three different chromosomal loci. They contain six extracellular cadherin domains, a transmembrane domain and a cytoplasmic tail differing from that of the classical cadherins [PCDH10 had been previously studied in PDAC primary tumors, but that study failed to find any correlation between PCDH10 methylation status and tumor staging [Protocadherins were included in the homophilic cell adhesion gene set that was shown to be subject to frequent alterations in an early study on transcriptome sequencing pancreatic cancers , but thiadherins . The cluadherins . Protocaadherins . The onsadherins . Promoteadherins , but in  staging .PCDH10, PCDHAC2 and PCDHGC5 that in our query of The Cancer Genome Atlas database resulted among the most frequently mutated in PDAC. Notably, PCDH10 promoter methylation had been previously suggested as a prognostic marker in prostate, gastric and colorectal cancer [PCDH10 methylation was identified as a factor associated with PDAC progression-free survival and, consequently, we suggest its possible role as a prognostic marker that might be useful for personalized treatment.Considering that protocadherins are frequently mutated in PDAC  and coull cancer . In our PCDH10 [PCDH10 mRNA expression analysis we used PCDH10 fully methylated pancreatic  and gastric (AGS) cancer cell lines, as well as PCDH10 unmethylated breast cancer cell line (MB-231) [Samples from surgically resected primary PDAC were collected from a series of 23 patients recruited at the Department of Surgery of “Casa Sollievo della Sofferenza” Hospital, IRCCS San Giovanni Rotondo. Only patients with histologically proven primary PDAC were enrolled in the study. Exclusion criteria for patients were a previous diagnosis for PDAC and neoadjuvant treatment before surgery. Tumors were staged in accordance with the TNM classification . ClinicaPCDH10 . For PCD(MB-231) , 17.TablResected PDACs and adjacent non-neoplastic tissues from the same patients were taken separately, immediately frozen in liquid nitrogen and stored at − 80 °C until the nucleic acid extraction. These control tissues were verified as tumor-free by a pathologist.Genomic DNA was isolated using the AllPrep DNA/RNA mini kit  according to the manufacturer’s instructions. DNA concentration and purity were controlled by NanoDrop Spectrophotometer .Bisulfite treatment was performed according to the manufacturer’s protocol . The bisulfite-treated DNA was amplified with primers designed according to MethPrimer . Primer BstUI restriction site were digested with BstUI (New England BioLabs) that recognizes the sequence 5′-CGCG-3′, retained in the bisulfite-treated methylated DNA, but not in the unmethylated DNA. The DNA digests were separated by denaturing high-performance liquid chromatography (dHPLC) .COBRA is a technique to semiquantitate the methylated and unmethylated DNA after sodium bisulfite treatment by using restriction enzyme cutting sites. PCR products containing CpG dinucleotides and at least 1 PCDHAC2 PCR product, encompassing the promoter region − 43 to + 192 bp from the transcription start site, provides two fragments of 210 and 25 bp, respectively; the 290 bp PCDHGC5 PCR product, encompassing the promoter region − 3287 to − 2997 bp upstream from the transcription start site, provides two fragments of 200 and 90 bp, respectively; the 196 bp PCDH10 PCR product, encompassing the promoter region − 1204 to − 1008 bp upstream from the transcription start site, has two cutting sites for BstUI and provides three fragments of 113, 52 and 31 base pairs, respectively. For PCDH10, the presence of two cutting sites for BstUI restriction enzyme hampered the interpretation of the analysis in case of partial methylation of the analyzed CpG islands. For this reason, we used BGS for this gene in all cases analyzed. Also for PCDHAC2 and PCDHGC5 DNA from a representative tumor and non-neoplastic sample were subjected to bisulfite genomic sequencing (BGS) to verify COBRA results independently.In case of methylated CpG dinucleotides, after enzymatic digestion, the 235 bp We directly sequenced the PCR products generated from bisulfite-treated templates with the same primers used for amplification Table .PCDH10, in cases showing CpG dinucleotides with the simultaneous presence of both peaks (T and C), we also analyzed data taking into account the relative height of the two peaks. The inclusion of this information in the analyses introduced marginal variations (2–3%) in the percentage of methylation status, and the subsequent analyses of the association between methylation and prognosis yielded virtually identical results.Sequencing analysis was performed using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Methylation status was expressed as the percentage of CpG methylated over the total number of CpG included in the sequence analyzed. In some cases, sequence analysis of bisulphite-treated DNA showed the simultaneous presence of both peaks (T and C), but in these cases there was always a major peak accounting for at least 70% of the total signal. This major peak was considered to call the island as methylated (C major peak) or unmethylated (T major peak) in subsequent analyses. For PCDH10 gene with previously published primers [PCDH10 cDNA RT-PCR amplified fragments were separated by dHPLC.Total RNA was extracted from Capan-2, AsPC-1, AGS, MB-231 cancer cell lines using Trizol reagent  was synthesised as previously described  and ampl primers . PCDH10 PCDH10 methylation levels according to the receiver operating characteristic (ROC) curve analysis. Consequently, the tumor was identified as PCDH10High with methylation levels above the cut-off threshold and PCDH10Low with methylation levels below the threshold. The relationships between PCDH10 methylation status and clinicopathological parameters were investigated by Pearson’s χ2 test.A cut-off of 52% was chosen to dichotomize PCDH10 methylation status with outcome was tested by Cox’s proportional hazards model. SPSS Version 15.0  was used for statistical analyses.Progression-free survival (PFS) was defined as the time from surgery to relapse, and overall survival (OS) as the time until death from any cause. Survival curves were plotted by the Kaplan-Meier method (log-rank test). Univariate analysis of http://david.abcc.ncifcrf.gov) to identify enriched biological themes and functional-related gene groups among the top 43 genes with > 10 mutations in PDAC according to TCGA. This analysis indicated that “Cadherins” including PCDH10, PCDHGC5, PCDH15, PCDHAC2, CDH10 were among functional-related gene groups statistically enriched after Bonferroni (P = 0.014) and Benjamini (P = 0.004) corrections . Based on the above results, in this study we analyzed CpG methylation for three genes that appeared to be frequently mutated in PDAC, including PCDHGC5 and PCDHAC2 that had not been studied before for epigenetic modifications and PCDH10, whose promoter methylation had been previously suggested as a prognostic marker in other cancers [To select genes that may play a key role in PDAC, we analyzed PDAC data in The Cancer Genome Atlas (TCGA) provisional database  to identify functionally related gene groups frequently mutated in this tumor. Protocadherins were among the most frequently mutated genes in PDAC samples analyzed by TCGA Table . We then cancers .Table 3TPCDHAC2, PCDHGC5 and PCDH10 was carried out in 11 pancreatic adenocarcinomas.An exploratory study of methylation analysis on PCDHAC2, dHPLC analysis of COBRA showed in all cases only the presence of the full-length fragment in tumors, indicating that the fragment was not cut because the C in the cutting site was unmethylated and thus converted to T by bisulfite treatment abolishing the cutting site  unmethylated for PCDH10, to assess whether methylation status of PCDH10 CpG dinucleotides was associated with effects on the expression of the corresponding transcript. In line with methylation status, Capan-2, AsPC-1 and AGS cell lines fully methylated for PCDH10 did not express the corresponding mRNA, whereas the cell line MB-231 unmethylated for PCDH10 expressed the corresponding transcript  and gastric (AGS) cancer cell lines fully methylated for , AsPC-1 PCDH10 methylation. In these cases, the percentages of methylation ranged from 8 to 91%, with a mean ± SE of 55.0 ± 7.8. The box-and-whisker diagram shows the PCDH10 methylation levels registered among 23 PDAC cases  tumors showed PCDH10 methylation status: tumors with methylation levels above 52% (n = 7) were considered PCDH10High, and those with methylation levels below the cut-off value were considered PCDH10Low (n = 16). By chi-square test, PCDH10 methylation status was found inversely correlated with the clinical presentation of jaundice (P = 0.036)  of those with PCDH10Low tumors. Death rates were 85.7 and 75.0% for patients with high and low methylation of PCDH10, respectively. At Kaplan–Meier analysis, PCDH10High was significantly associated with worse PFS rates (P = 0.008), but not with OS  of patients with  OS Fig. .Fig. 4KaPCDH10High was a prognostic factor influencing PFS , but not the OS , no additional prognostic factors are recommended for clinical care of PDAC patients. Thus, additional prognostic biomarkers are needed to provide a better risk assessment.KRAS, CDKN2A, TP53, SMAD4), mutations in SMAD4 were associated with poor survival, whereas mutations in KRAS, CDKN2A and TP53, or the presence of multiple (> 4) mutations or homozygous deletions among the most frequently mutated genes were not associated with survival [Recent studies showed that PDAC is not a clinically homogeneous disease, but molecularly defined subsets of patients with distinct clinical features, including prognosis and response to therapy, can be identified by integrated genome-wide analyses –6. Amongsurvival .reprimo, a gene involved in p53-induced G2 cell cycle arrest, was shown to associate with worse prognosis [In addition to mutations, epigenetic modifications may play an important role in PDAC as suggested by the observation that aberrant CpG island methylation of rognosis . Howeverrognosis .Since genes that are frequently mutated in PDAC may be crucial for the biology of this tumor, and they might also be a target of epigenetic dysregulation, we searched for genes frequently mutated in PDAC by querying The Cancer Genome Atlas (TCGA) provisional database. The bioinformatics analysis identified protocadherins among the most mutated genes in PDAC.PCDHAC2, PCDHGC5 and PCDH10 because they emerged among the most mutated genes in PDAC through the aforementioned unbiased in silico approach. Notably, the methylation status of PCDHAC2 and PCDHGC5 were never analyzed before in PDAC, while PCDH10 had been previously studied in PDAC cancer cell lines [Therefore, we evaluated whether the epigenetic differences in terms of promoter methylation of protocadherins between the tumor and non-tumor tissue samples could be used as survival predictors in PDAC patients. In particular, we studied the promoter methylation of ll lines  and one ll lines .PCDHAC2 resulted hypomethylated, whereas PCDHGC5 was hypermethylated in all PDAC samples and the same patterns of methylation were also observed in matched adjacent non-neoplastic pancreatic tissues, suggesting that CpG promoter methylation of these genes does not play a major role in the biology of this tumor. Conversely, PCDH10, that resulted unmethylated in adjacent non-neoplastic pancreatic tissues showed a variable degree of methylation ranging from high to low levels in matched PDAC samples. As expected, PCDH10 methylation status correlated with the lack of expression of the corresponding transcript in PCDH10 fully methylated cancer cell lines and, conversely, with expression of PCDH10 in the unmethylated cell line analyzed. In line with our findings, a previous study [PCDH10 methylation and loss of PCDH10 mRNA expression in pancreatic, gastric and colorectal cancers tissues.In our study us study  found a PCDH10 methylation among patients led us to investigate the possible correlations between CpG dinucleotide methylation in this gene and PDAC clinical outcome. In this analysis we found, for the first time, an association between PCDH10 promoter methylation status and PDAC patients outcomes, being the hypermethylation of the gene associated with shorter progression-free survival.The variability of PCDH10High rather than PCDH10Low tumors . However, possibly because of the high rates of death, the relatively small differences among cohorts and the limited number of patients analyzed, we did not find any correlation between PCDH10 status and overall survival.Deaths occurred at high rates in both cohorts of PDAC patients and the percentage tended to be higher among PDAC patients with PCDH10 was already reported to be inactivated by promoter methylation in various types of cancer, including non-small cell lung cancer [PCDH10 as an oncosuppressor gene, re-expression of this gene by transfection in a gastric cancer cell line inhibited the proliferation, migration, invasion ability, as well as its tumor growth in mice [PCDH10 was associated with poor prognosis in patients with gastric cancer [PCDH10 represents an adverse prognostic marker for the survival of patients with CRC [PCDH10 as oncosuppressor gene in PDAC was investigated only in pancreatic cancer cell lines where this gene was silenced by methylation and its re-expression by transfection inhibited the proliferation, migration, invasion ability and induced apoptosis [PCDH10 methylation in pancreatic tumor samples failed to find any correlation between PCDH10 methylation status and PDAC staging, which was the pathologic feature analyzed in that study [g cancer , gastricg cancer , colorecg cancer , nasophag cancer , endometg cancer , 27, blag cancer , cervicag cancer , suggest in mice . Furtherc cancer . In linewith CRC . In pancpoptosis . The onlat study . Also inPromoter methylation has been reported as a promising predictive biomarker in many human cancers. However, a better understanding of the specific epigenetic changes affecting the prognosis of PDAC is necessary.PCDH10 can predict the patients’ prognosis and may have a significant impact on the outcome in terms of progression-free survival of the patients with PDAC. In particular, high levels of PCDH10 promoter methylation could be useful to identify patients at high risk of disease progression and early death after surgical treatment, contributing to a more accurate stratification of PDAC patients for personalized clinical management.In our study, we identified for the first time that methylation status of


A paucity of advances in the development of novel therapeutic agents for squamous cell carcinomas of the head and neck, oral cavity (OSCC) and oropharynx, has stagnated disease free survival rates over the past two decades. Although immunotherapies targeted against checkpoint inhibitors such as PD-1 or CTLA-4 are just now entering the clinic for late stage disease with regularity the median improvement in overall survival is only about three months. There is an urgent unmet clinical need to identify new therapies that can be used alone or in combination with current approaches to increase survival by more than a few months. Activation of the apoptotic arm of the unfolded response (UPR) with small molecules and natural products has recently been demonstrated to be a productive approach in pre-clinical models of OSCC and several other cancers. The aim of current study was to perform a high throughput screen (HTS) with a diverse chemical library to identify compounds that could induce CHOP, a component of the apoptotic arm of the UPR. Disulfiram  the well-known aversion therapy used to treat chronic alcoholism emerged as a hit that could generate reactive oxygen species, activate the UPR and apoptosis and reduce proliferation in OSCC cell cultures and xenografts. A panel of murine embryonic fibroblasts null for key UPR intermediates  was resistant to DSF suggesting that an intact UPR is a key element of the mechanism regulating the antiproliferative effects of DSF. The Oral Cancer Foundation (OCF) has accurately predicted the annual incidence of oral (OSCC) and oropharyngeal cancer in the United States since 2002. OCF anticipates that 2019 will see 50,000 new cases with about 10,000 deaths, substantially higher than in 2000 when the expected number was ~30,000 and had been unchanged for the previous two decades ,5. The bThe high morbidity and mortality associated with OSCC are bolstered by the fact that approximately 60% of patients present with late stage disease. The primary modes of treatment for OSCC remain in the purview of surgeons and radiation oncologists as the addition of chemotherapy and biological agents have provided only modest improvements in the last three decades. Promising clinical trials in 2016 and 2017 saw the introduction of immune modulating checkpoint inhibitors to the arsenal of FDA approved compounds for late stage and recurrent head and neck cancer however, these compounds are not yet approved for OSCC and there is an urgent unmet clinical need to find new approaches for patients afflicted with this devastating disease. Our group has previously hypothesized that targeting the unfolded protein response (UPR), to increase nuclear pools of C/ebp homologous protein (CHOP), might specifically leverage the highly secretory nature of OSCC towards its demise ,10,11. EmRNA molecules are translated into proteins with different efficiencies depending on the length and complexity of the structure of their 5’ untranslated regions. Many oncogenes have long highly unstructured 5’ UTRs that are not conveniently accessed by translation initiation factors, which keeps their translation at low levels during homeostasis. It is now clear that eukaryotic translation initiation factor 2, (eIF2α) and eIF4E are mis-regulated in a subset of acute ,13 and cXBP1) to produce a potent basic leucine zipper transcription factor that increases the expression of ER chaperones and proteins that enhance ER-associated degradation (ERAD) [The UPR is a remedy to perturbations in the secretory pathway that disrupt protein folding, post-translational modification, or trafficking out of the ER. The UPR is orchestrated by a tripartite signaling conduit characterized by a transient halt in general protein translation and increased expression of folding-associated enzymes . The ER n (ERAD) ,23. If tn (ERAD) .As CHOP is known to be a mediator of ER stress-induced apoptosis, a cell-based high throughput screen (HTS) was undertaken to identify small molecule activators . We hypoOne of the agents identified in the HTS screen was tetraethylthiuram disulfide, also known as disulfiram (DSF), that has been used safely and frequently at large oral doses for the treatment of alcoholism since 1948. DSF and various metabolites are potent inhibitors of mitochondrial and cytosolic aldehyde dehydrogenases (ALDH) ,27. DSF A253 and Cal27 cells were obtained from ATCC; HN6, HN12, and HN30 cells were provided by Dr. George Yoo at Karmanos Cancer Institute, and STR-validated UMSCC-1, -14A and -23 cells were from Dr. Tom Carey at the University of Michigan. All cancer cell lines were maintained under standard tissue culture conditions in DMEM supplemented with 10% FBS and antibiotics as previously described . Normal DSF and NSC-1771 were obtained from MicroSource Discovery Inc.  or Acros Organics . 100 mM solutions were prepared in DMSO and stored at −20 °C. Approximately 60,000 small molecules and natural product extracts were screened at the University of Michigan Center for Chemical Genomics as previously described .To detect oxidized proteins in lysates from OSCC cells treated with DSF, the Protein Carbonyl Colorimetric Assay Kit from Caymen  was used according to the manufacturer’s protocol. 5 SCC-14A cells were plated in triplicate the day before treatment with 10 μM DSF or 0.5 μM thapsigargin (Tg). At harvest, cells were rinsed twice with PBS and 5 μL 35S-Met was added and incubated for 15 min at 37 °C. 35S-Met medium was removed and plates were placed on ice and washed twice with cold PBS. Whole cell lysates were prepared in 200 μL RIPA with PMSF without protease inhibitors. 5 μL of each sample in 5 mL of scintillation fluid was used to measure cpm; values were normalized to total proteins assay by the Lowry method [5.5 × 10y method .Cell Titer Glo, an ATP-based luminescent proliferation assay from Promega , was used as a relative measure of viable cells. Trypan-blue exclusion assays were employed to determine the ratio of live:dead cells. Whole-cell lysates were prepared in modified radioimmunoprecipitation (RIPA) buffer with PMSF and protease inhibitors. Briefly, 50–80 micrograms of cell lysate was resolved on 7.5% or 12.5% SDS-PAGE gels and electrically transferred to PVDF membranes . Membranes were probed overnight at 4 °C with antibodies for caspases 3, caspase 9, GRP78/BIP, CHOP and phosphorylated eIF2α ; ATF4 , as previously described . FollowiXBP1 was performed with a single human-specific primer pair ACA CGC TTG GGA ATG GAC AC and CCA TGG GAA GAT GTT CTG GG [18S (Hs99999901_s1), CHOP/DDIT3 (Hs01090850_m1), GADD34/PPP1R15 (Hs00169585_m1), ATF3 (Hs00910173_m1), ATF4 (Hs00909569_g1), XBP1s (Hs00231936_m1), and GRP78/BiP/HSPA5 (Hs99999174_m1). All experiments were performed with triplicate samples and each measured at least three times; data are represented as standard error of the mean. mRNA was harvested from DSF treated UMSCC-1 and -14A cells using Cells to CT™  as previously described . Semi-quT CTG GG . RT-qPCR6 SCC23-CHOP-luc cells were injected subcutaneously in the flanks of 6–12 week old female athymic SCID mice and allowed to grow until palpable solid masses were appreciable (~3 mM in diameter). Mice were randomized to 100 mg/kg DSF or carrier  daily delivered by oral gavage. Xenograft volume was calculated using ½ (L X W2), where length and width are the widest. Daily oral gavage was continued until tumors exceeded 1cm2 or animals became moribund as per WSU IACUC protocol. Bioluminescent images of CHOP-luciferase expressing cells were captured 24 h after DSF or vehicle administration; random mice were IP injected with 100 mg/kg luciferin and imaged on a Kodak In-Vivo Extreme. All animal studies were performed in accordance withthe Wayne State University IACUC (protocol ID 16-07-111). 1035S incorporation, carbonyl formation, etc.). In each case the level of confidence was represented as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.Each data set represented graphically or mentioned in the text was analyzed by a referee uninvolved with the experimental procedure. Two-way ANOVA was used to appreciate differences in proliferation between wildtype and knockout murine embryonic fibroblasts (MEF). Two-tailed tests were used to appreciate differences between treated and untreated cell cultures  strategy used to discover small molecules and natural products that activated the apoptotic arm of the UPR [ the UPR . The ass the UPR  however, the UPR .DSF induces ER stress and the UPR in OSCC cell lines. To elucidate the ability of DSF to activate the UPR, SCC-1 and SCC-14A cells were treated for six hours. RT-qPCR revealed dose-dependent increases of transcripts for UPR markers  precipitation and radioanalysis confirmed 10 μM DSF could rapidly and significantly reduce protein synthesis , and phosphorylation resistant eIF2α Ser51Ala mutant (A/A) MEFs were much more sensitive to the antiproliferative effects of DSF than wildtype (S/S)  H, consise assays . ConsideDSF induced reactive oxygen species (ROS) are required for UPR induction in OSCC. DSF is well-known and potent redox modulator, and ROS generation is a well-characterized stimulus of ER stress. To more precisely elucidate the mechanism by which DSF induced ER stress and the UPR, OSCC cells were treated with 10 μM DSF for 24 h in the presence of the potent superoxide-specific scavenger tiron (Ti). ATP-based luminescent proliferation assays  revealed CHOP-luc activity reporter was apparent in DSF treated mice after 24 h  agents used in the production of rubber . WilliamThe anticancer effects of DSF have been studied in many pre-clinical cancer models including brain , glioblaThe cancer stem cell (CSC) hypothesis is rooted in the idea that tumor proliferation, metastasis, resistance to therapy and recurrence are driven by a small subset of progenitor cells. CSC’s are characterized by the ability to reproduce progenitor cells  and to divide into daughter cells that populate tumors . AlthougChop or Atf4 required much higher concentrations of DSF to reduce proliferation than wildtype littermate controls in a time and concentration dependent manner, indicating an intact PERK arm of the UPR is required for efficient DSF-mediated apoptosis. The aberrant proliferation and secretory nature of malignant cells are dependent on the uninterrupted manufacture of de novo peptides. When protein folding demands outstrip the ability of the ER to fold and post-translationally modify new polypeptides the UPR is activated to restore homeostasis or eradicate the cell. Increased expression of ER stress markers and eukaryotic translation initiation factors in cancers of the head and neck, oral cavity and thyroid have been widely reported ,17,64,652−) formation occurs at the outer mitochondrial membrane, on both sides of the inner mitochondrial membrane and in the matrix, and can traverse to the cytosol freely when a mitochondrion is damaged or through voltage-dependent anion channels is response to various stimuli [Malignant cells generally have higher ROS activity than non-malignant cells  and it w stimuli . Superox stimuli . This ob stimuli .Chop promoter, whereas expression of luciferase in the Xbp1 reporter relied on the splicing of the 26 base intron and recombination of the firefly luciferase mRNA. XBP1 spicing is currently regarded as a hallmark of ER stress, as IRE1α is the only known exoribonuclease able to remove the 26 base intron that holds XBP1 in an inactive state. It is possible that the ability of DSF to splice XBP1 might contribute to the cancer cell selectivity. Tumor-adjacent normal cells with no or low basal UPR activation might be able to mount an effective adaptive UPR, overcome the stress, and return to homeostasis. Malignant cells on the other hand, become overwhelmed by the increased stress, and are shunted toward UPR (CHOP)-mediated apoptosis.Considering the HTS reporter data , where C2+ or Cu2+; and (ii) in early stage preclinical models . An effective approach for the use of DSF in OSCC may be to treat patients with long term metronomic dosing after curative or salvage surgery in combination with a divalent cation supplement . Since chronic alcohol consumption is a feature of many head and neck cancers DSF might provide an additional advantage to OSCC patients. With a five-year survival rate as low as 20% for patients with distant metastases from the floor of mouth, OSCC prognoses are stagnated amongst the worst of any cancer. The anti-alcohol and anti-tumor effects of DSF might provide a much needed boost in quality of life and survival.The successful use of DSF in preclinical models of cancer has been most encouraging when: (i) used in combination (complexed or supplemented) with divalent cations such as Zn


Neuropsychiatric secondary symptoms and altered physical function are prevalent among persons with Alzheimer’s Disease and related dementia disorders (ADRD) which increase healthcare costs and caregiver burden. Adapted dance is a promising intervention that may improve these symptoms and physical function. The purpose of this study is to test whether 12 weeks of adapted dance (60 min 2x/week) improves agitation, physical function, and reduces caregiver burden. An experimental crossover design will be used. ADRD residents with a Montreal Cognitive Assessment score between 6-26, Timed up and go of <20 seconds, Cohen-Mansfield Agitation Inventory Score (CMAI) of >15, and do not use oxygen or assistive device will be eligible to participate. Outcomes will be assessed at baseline and every 4 weeks during each study arm for 24 weeks. Measures include CMAI and the Neuropsychiatric Inventory-Clinician Scale for agitation; Short Physical Performance Battery for physical function; and Zarit Burden Interview for caregiver burden.