Morphology-guided transcriptomic analysis of human pancreatic cancer organoids reveals microenvironmental signals that enhance invasion.

Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared with matched noninvasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified 3 distinct transcriptomic groups in invasive organoids, 2 of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGF-ß1, IL-6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.

Ganglion cyst at the proximal tibiofibular joint - A rare cause of compression neuropathy of the peroneal nerve.

Ganglion cysts are fluid filled sacs which develop near joints and tendons and are usually asymptomatic. Lower limb ganglion cysts are rare occurrences especially those situated around joint spaces causing nerve compression. We present the case of a 68 year-old female with history of progressive swelling in the left antero-lateral leg, associated with pain, and neurological symptoms of peroneal nerve compression. Magnetic resonance imaging (MRI) revealed a large proximal tibiofibular joint ganglion cyst causing peroneal nerve compression. One year following the left sided presentation, the patient presented with similar but less severe symptoms in her right antero-lateral leg. MRI revealed a small juxta-articular ganglion cyst in the right proximal tibiofibular joint space. We discuss etiology, symptoms, and management of lower limb ganglion cysts.

Reciprocal interplay between asporin and decorin: Implications in gastric cancer prognosis.

Effective patient prognosis necessitates identification of novel tumor promoting drivers of gastric cancer (GC) which contribute to worsened conditions by analysing TCGA-gastric adenocarcinoma dataset. Small leucine-rich proteoglycans, asporin (ASPN) and decorin (DCN), play overlapping roles in development and diseases; however, the mechanisms underlying their interplay remain elusive. Here, we investigated the complex interplay of asporin, decorin and their interaction with TGFß in GC tumor and corresponding normal tissues. The mRNA levels, protein expressions and cellular localizations of ASPN and DCN were analyzed using real-time PCR, western blot and immunohistochemistry, respectively. The protein-protein interaction was predicted by in-silico interaction analysis and validated by co-immunoprecipitation assay. The correlations between ASPN and EMT proteins, VEGF and collagen were achieved using western blot analysis. A significant increase in expression of ASPN in tumor tissue vs. normal tissue was observed in both TCGA and our patient cohort. DCN, an effective inhibitor of the TGFß pathway, was negatively correlated with stages of GC. Co-immunoprecipitation demonstrated that DCN binds with TGFß, in normal gastric epithelium, whereas in GC, ASPN preferentially binds TGFß. Possible activation of the canonical TGFß pathway by phosphorylation of SMAD2 in tumor tissues suggests its role as an intracellular tumor promoter. Furthermore, tissues expressing ASPN showed unregulated EMT signalling. Our study uncovers ASPN as a GC-promoting gene and DCN as tumor suppressor, suggesting that ASPN can act as a prognostic marker in GC. For the first time, we describe the physical interaction of TGFß with ASPN in GC and DCN with TGFß in GC and normal gastric epithelium respectively. This study suggests that prevention of ASPN-TGFß interaction or overexpression of DCN could serve as promising therapeutic strategies for GC patients.

Adeno-associated virus in the liver: natural history and consequences in tumour development.

OBJECTIVE: Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterise the natural history of AAV infection in the liver and its consequence in tumour development. DESIGN: Viral DNA was quantified in tumour and non-tumour liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analysed using a DNAse/TaqMan-based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. RESULTS: AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site. CONCLUSION: We provided an integrated analysis of the wild-type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. Clonal AAV insertions were positive selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus.

Liquid biopsy: miRNA as a potential biomarker in oral cancer.

Oral cancer is one of the leading cancers in South-Asian countries. Despite the easy access of the oral cavity, the detection and five year survival rates of OSCC patients are dismal. Identification of non-invasive biomarkers to determine the progression and recurrence of OSCC could be of immense help to patients. Recent studies on oral cancer suggest the importance of non-invasive biomarker development. Micro-RNAs (miRNAs) are one of the important components of the cell-free nucleic acids available in different body fluids. Here, we have reviewed the current understanding of circulating miRNAs as non-invasive biomarkers in different body fluids of oral cancer patients. A number of circulating miRNAs are found to be common in the body fluids of OSCC patients, while many of these are study specific, the possible sources of this variability could be due to differences in sample processing, assay procedure, clinical stage of the disease, oral habit and environmental factors. The prognostic and therapeutic significance of these circulating miRNAs are suggested by several studies. Mir-371, mir-150, mir-21 and mir-7d were found to be potential prognostic markers, while mir-134, mir-146a, mir-338 and mir-371 were associated with metastases. The prognostic markers, mir-21 and mir-7d were also found to be significantly correlated with resistance to chemotherapy, while mir-375, mir-196 and mir-125b were significantly correlated with sensitivity to radiotherapy. Despite the promising roles of circulating miRNAs, challenges still remain in unravelling the exact regulation of these miRNAs before using them for targeted therapy.

Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma.

Adeno-associated virus type 2 (AAV2) is a defective DNA virus that was previously considered to be non-pathogenic. We identified somatic AAV2 integration in a subset of 11 hepatocellular carcinomas (HCC) that mainly developed in normal liver without known etiology through recurrent insertional mutagenesis in cancer driver genes such as telomerase reverse transcriptase (TERT), cyclin A2 (CCNA2), cyclin E1 (CCNE1), tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10), and lysine (K)-specific methyltransferase 2B (KMT2B).

Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.

Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC.

A genome-wide search for non-UGT1A1 markers associated with unconjugated bilirubin level reveals significant association with a polymorphic marker near a gene of the nucleoporin family.

Variants in the UGT1A1 gene and its promoter are known to determine levels of unconjugated bilirubin (UCB), but do not explain all cases of unconjugated hyperbilirubinemia. To discover associations with variants in genes other than UGT1A1, we undertook a genome-wide association study. We recruited 200 participants to cover the entire range of quantitative variation in UCB level. The data set -- after data curation, including analyses for population stratification and cryptic relatedness -- comprised genotypes at 512,349 SNP loci on 182 individuals. Quantitative trait locus (QTL) association analyses were performed, after adjusting the UCB level for effects of age, gender, and genotype at the dinucleotide (TA) insertion locus in UGT1A1 that is known to significantly modulate UCB level. A significant association of a polymorphic marker (rs2328136) near the NUP153 gene (which produces a 153 kDa nucleoporin) was obtained (p = 0.002, after multiple-testing correction). The frequency of the variant allele (A) at the rs2328136 locus in our study population is 40%, higher than most global populations. NUP153, whose product is a major regulatory factor in bidirectional transport of biomolecules across nucleus to cytosol, is associated with the transport of biliverdin reductase, which is important for bilirubin conjugation.

Patterns of skeletal fractures in child abuse: systematic review.

OBJECTIVES: To systematically review published studies to identify the characteristics that distinguish fractures in children resulting from abuse and those not resulting from abuse, and to calculate a probability of abuse for individual fracture types. DESIGN: Systematic review. DATA SOURCES: All language literature search of Medline, Medline in Process, Embase, Assia, Caredata, Child Data, CINAHL, ISI Proceedings, Sciences Citation, Social Science Citation Index, SIGLE, Scopus, TRIP, and Social Care Online for original study articles, references, textbooks, and conference abstracts until May 2007. STUDY SELECTION: Comparative studies of fracture at different bony sites, sustained in physical abuse and from other causes in children <18 years old were included. Review articles, expert opinion, postmortem studies, and studies in adults were excluded. Data extraction and synthesis Each study had two independent reviews (three if disputed) by specialist reviewers including paediatricians, paediatric radiologists, orthopaedic surgeons, and named nurses in child protection. Each study was critically appraised by using data extraction sheets, critical appraisal forms, and evidence sheets based on NHS Centre for Reviews and Dissemination guidance. Meta-analysis was done where possible. A random effects model was fitted to account for the heterogeneity between studies. RESULTS: In total, 32 studies were included. Fractures resulting from abuse were recorded throughout the skeletal system, most commonly in infants (<1 year) and toddlers (between 1 and 3 years old). Multiple fractures were more common in cases of abuse. Once major trauma was excluded, rib fractures had the highest probability for abuse (0.71, 95% confidence interval 0.42 to 0.91). The probability of abuse given a humeral fracture lay between 0.48 (0.06 to 0.94) and 0.54 (0.20 to 0.88), depending on the definition of abuse used. Analysis of fracture type showed that supracondylar humeral fractures were less likely to be inflicted. For femoral fractures, the probability was between 0.28 (0.15 to 0.44) and 0.43 (0.32 to 0.54), depending on the definition of abuse used, and the developmental stage of the child was an important discriminator. The probability for skull fractures was 0.30 (0.19 to 0.46); the most common fractures in abuse and non-abuse were linear fractures. Insufficient comparative studies were available to allow calculation of a probability of abuse for other fracture types. CONCLUSION: When infants and toddlers present with a fracture in the absence of a confirmed cause, physical abuse should be considered as a potential cause. No fracture, on its own, can distinguish an abusive from a non-abusive cause. During the assessment of individual fractures, the site, fracture type, and developmental stage of the child can help to determine the likelihood of abuse. The number of high quality comparative research studies in this field is limited, and further prospective epidemiology is indicated.