Can upfront DPYD extended variant testing reduce toxicity and associated hospital costs of fluoropyrimidine chemotherapy? A propensity score matched analysis of 2022 UK patients.

AIM: To independently assess the impact of mandatory testing using an extended DPYD variant panel (ToxNav®) and consequent dose adjustment of Capecitabine/5-FU on recorded quantitative toxicity, symptoms of depression, and hospital costs. METHODS: We used propensity score matching (PSM) to match 466 patients tested with ToxNav® with 1556 patients from a historical cohort, and performed regression analysis to estimate the impact of ToxNav®on toxicity, depression, and hospital costs. RESULTS: ToxNav® appeared to reduce the likelihood of experiencing moderate (OR: 0.59; 95%CI: 0.45-0.77) and severe anaemia (OR: 0.55; 95%CI: 0.33-0.90), and experience of pain for more than 4 days a week (OR: 0.50; 95%CI: 0.30-0.83), while it increased the likelihood of mild neutropenia (OR: 1.73; 95%CI: 1.27-2.35). It also reduced the cost of chemotherapy by 12% (95%CI: 3-31) or £9765, the cost of non-elective hospitalisation by 23% (95%CI: 8-36) or £2331, and the cost of critical care by 21% (95%CI: 2-36) or £1219 per patient. For the DPYD variant associated with critical risk of toxicity (rs3918290), the improved non-elective hospital costs were > £20,000, whereas variants associated with hand-foot syndrome toxicity had no detectable cost improvement. CONCLUSION: Upfront testing of DPYD variants appears to reduce the toxicity burden of Capecitabine and 5-FU in cancer patients and can lead to substantial hospital cost savings, only if the dose management of the drugs in response to variants detected is standardised and regulated.

New indicators and indexes for benchmarking university-industry-government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project.

BACKGROUND: While the European Union is striving to become the 'Innovation Union', there remains a lack of quantifiable indicators to compare and benchmark regional innovation clusters. To address this issue, a HealthTIES (Healthcare, Technology and Innovation for Economic Success) consortium was funded by the European Union's Regions of Knowledge initiative, research and innovation funding programme FP7. HealthTIES examined whether the health technology innovation cycle was functioning differently in five European regional innovation clusters and proposed regional and joint actions to improve their performance. The clusters included BioCat (Barcelona, Catalonia, Spain), Medical Delta (Leiden, Rotterdam and Delft, South Holland, Netherlands), Oxford and Thames Valley (United Kingdom), Life Science Zürich (Switzerland), and Innova Észak-Alföld (Debrecen, Hungary). METHODS: Appreciation of the 'triple helix' of university-industry-government innovation provided the impetus for the development of two quantifiable innovation indexes and related indicators. The HealthTIES H-index is calculated for disease and technology platforms based on the h-index proposed by Hirsch. The HealthTIES Innovation Index is calculated for regions based on 32 relevant quantitative and discriminative indicators grouped into 12 categories and 3 innovation phases, namely 'Input' (n = 12), 'Innovation System' (n = 9) and 'Output' (n = 11). RESULTS: The HealthTIES regions had developed relatively similar disease and technology platform profiles, yet with distinctive strengths and weaknesses. The regional profiles of the innovation cycle in each of the three phases were surprisingly divergent. Comparative assessments based on the indicators and indexes helped identify and share best practice and inform regional and joint action plans to strengthen the competitiveness of the HealthTIES regions. CONCLUSION: The HealthTIES indicators and indexes provide useful practical tools for the measurement and benchmarking of university-industry-government innovation in European medical and life science clusters. They are validated internally within the HealthTIES consortium and appear to have a degree of external prima facie validity. Potentially, the tools and accompanying analyses can be used beyond the HealthTIES consortium to inform other regional governments, researchers and, possibly, large companies searching for their next location, analyse and benchmark 'triple helix' dynamics within their own networks over time, and to develop integrated public-private and cross-regional research and innovation strategies in Europe and beyond.

A germline mutation of CDKN2A and a novel RPLP1-C19MC fusion detected in a rare melanotic neuroectodermal tumor of infancy: a case report.

BACKGROUND: Melanotic neuroectodermal tumor of infancy (MNTI) is exceptionally rare and occurs predominantly in the head and neck (92.8 % cases). The patient reported here is only the eighth case of MNTI presenting in an extremity, and the first reported in the fibula. CASE PRESENTATION: A 2-month-old female presented with a mass arising in the fibula. Exhaustive genomic, transcriptomic, epigenetic and pathological characterization was performed on the excised primary tumor and a derived cell line. Whole-exome analysis of genomic DNA from both the tumor and blood indicated no somatic, non-synonymous coding mutations within the tumor, but a heterozygous, unique germline, loss of function mutation in CDKN2A (p16(INK4A), D74A). SNP-array CGH on DNA samples revealed the tumor to be euploid, with no detectable gene copy number variants. Multiple chromosomal translocations were identified by RNA-Seq, and fusion genes included RPLP1-C19MC, potentially deregulating the C19MC cluster, an imprinted locus containing microRNA genes reactivated by gene fusion in embryonal tumors with multilayered rosettes. Since the presumed cell of origin of MNTI is from the neural crest, we also compared gene expression with a dataset from human neural crest cells and identified 185 genes with significantly different expression. Consistent with the melanotic phenotype of the tumor, elevated expression of tyrosinase was observed. Other highly expressed genes encoded muscle proteins and modulators of the extracellular matrix. A derived MNTI cell line was sensitive to inhibitors of lysine demethylase, but not to compounds targeting other epigenetic regulators. CONCLUSIONS: In the absence of somatic copy number variations or mutations, the fully transformed phenotype of the MNTI may have arisen in infancy because of the combined effects of a germline CDKN2A mutation, tumor promoting somatic fusion genes and epigenetic deregulation. Very little is known about the etiology of MNTI and this report advances knowledge of these rare tumors by providing the first comprehensive genomic, transcriptomic and epigenetic characterization of a case.

IGF signaling directs ventricular cardiomyocyte proliferation during embryonic heart development.

Secreted factors from the epicardium are believed to be important in directing heart ventricular cardiomyocyte proliferation and morphogenesis, although the specific factors involved have not been identified or characterized adequately. We found that IGF2 is the most prominent mitogen made by primary mouse embryonic epicardial cells and by a newly derived immortalized mouse embryonic epicardial cell line called MEC1. In vivo, Igf2 is expressed in the embryonic mouse epicardium during midgestation heart development. Using a whole embryo culture assay in the presence of inhibitors, we confirmed that IGF signaling is required to activate the ERK proliferation pathway in the developing heart, and that the epicardium is required for this response. Global disruption of the Igf2 gene, or conditional disruption of the two IGF receptor genes Igf1r and Insr together in the myocardium, each resulted in a significant decrease in ventricular wall proliferation and in ventricular wall hypoplasia. Ventricular cardiomyocyte proliferation in mutant embryos was restored to normal at E14.5, concurrent with the establishment of coronary circulation. Our results define IGF2 as a previously unexplored epicardial mitogen that is required for normal ventricular chamber development.

IR/IGF1R signaling as potential target for treatment of high-grade osteosarcoma.

BACKGROUND: High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment. METHODS: We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma - mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays. RESULTS: OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with IC50s below 100 nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines. CONCLUSIONS: This study provides an in vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma.

Structure and functional analysis of the IGF-II/IGF2R interaction.

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.

Structure of the Human Cation-Independent Mannose 6-Phosphate/IGF2 Receptor Domains 7-11 Uncovers the Mannose 6-Phosphate Binding Site of Domain 9.

The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7-10 within two multi-domain constructs, D9-10 and D7-11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7-11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7-11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.

Structural insights into the interaction of insulin-like growth factor 2 with IGF2R domain 11.

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.

Maternal transmission of an Igf2r domain 11: IGF2 binding mutant allele (Igf2rI1565A) results in partial lethality, overgrowth and intestinal adenoma progression.

The cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R or IGF2R) traffics IGF2 and M6P ligands between pre-lysosomal and extra-cellular compartments. Specific IGF2 and M6P high-affinity binding occurs via domain-11 and domains-3-5-9, respectively. Mammalian maternal Igf2r allele expression exceeds the paternal allele due to imprinting (silencing). Igf2r null-allele maternal transmission results in placenta and heart over-growth and perinatal lethality (>90%) due to raised extra-cellular IGF2 secondary to impaired ligand clearance. It remains unknown if the phenotype is due to either ligand alone, or to both ligands. Here, we evaluate Igf2r specific loss-of-function of the domain-11 IGF2 binding site by replacing isoleucine with alanine in the CD loop (exon 34, I1565A), a mutation also detected in cancers. Igf2rI1565A/+p maternal transmission (heterozygote), resulted in placental and embryonic over-growth with reduced neonatal lethality (<60%), and long-term survival. The perinatal mortality (>80%) observed in homozygotes (Igf2rI1565A/I1565A) suggested that wild-type paternal allele expression attenuates the heterozygote phenotype. To evaluate Igf2r tumour suppressor function, we utilised intestinal adenoma models known to be Igf2 dependent. Bi-allelic Igf2r expression suppressed intestinal adenoma (ApcMin). Igf2rI1565A/+p in a conditional model (Lgr5-Cre, Apcloxp/loxp) resulted in worse survival and increased adenoma proliferation. Growth, survival and intestinal adenoma appear dependent on IGF2R-domain-11 IGF2 binding.

Soluble IGF2 receptor rescues Apc(Min/+) intestinal adenoma progression induced by Igf2 loss of imprinting.

The potent growth-promoting activity of insulin-like growth factor-II (IGF-II) is highly regulated during development but frequently up-regulated in tumors. Increased expression of the normally monoallelic (paternally expressed) mouse (Igf2) and human (IGF2) genes modify progression of intestinal adenoma in the Apc(Min/+) mouse and correlate with a high relative risk of human colorectal cancer susceptibility, respectively. We examined the functional consequence of Igf2 allelic dosage (null, monoallelic, and biallelic) on intestinal adenoma development in the Apc(Min/+) by breeding with mice with either disruption of Igf2 paternal allele or H19 maternal allele and used these models to evaluate an IGF-II-specific therapeutic intervention. Increased allelic Igf2 expression led to elongation of intestinal crypts, increased adenoma growth independent of systemic growth, and increased adenoma nuclear beta-catenin staining. By introducing a transgene expressing a soluble form of the full-length IGF-II/mannose 6-phosphate receptor (sIGF2R) in the intestine, which acts as a specific inhibitor of IGF-II ligand bioavailability (ligand trap), we show rescue of the Igf2-dependent intestinal and adenoma phenotype. This evidence shows the functional potency of allelic dosage of an epigenetically regulated gene in cancer and supports the application of an IGF-II ligand-specific therapeutic intervention in colorectal cancer.

Retrospective audit of 957 consecutive 18F-FDG PET-CT scans compared to CT and MRI in 493 patients with different histological subtypes of bone and soft tissue sarcoma.

BACKGROUND: The use of 18F-FDG PET-CT (PET-CT) is widespread in many cancer types compared to sarcoma. We report a large retrospective audit of PET-CT in bone and soft tissue sarcoma with varied grade in a single multi-disciplinary centre. We also sought to answer three questions. Firstly, the correlation between sarcoma sub-type and grade with 18FDG SUVmax, secondly, the practical uses of PET-CT in the clinical setting of staging (during initial diagnosis), restaging (new baseline prior to definitive intervention) and treatment response. Finally, we also attempted to evaluate the potential additional benefit of PET-CT over concurrent conventional CT and MRI. METHODS: A total of 957 consecutive PET-CT scans were performed in a single supra-regional centre in 493 sarcoma patients (excluding GIST) between 2007 and 2014. We compared, PET-CT SUVmax values in relation to histology and FNCCC grading. We compared PET-CT findings relative to concurrent conventional imaging (MRI and CT) in staging, restaging and treatment responses. RESULTS: High-grade (II/III) bone and soft tissue sarcoma correlated with high SUVmax, especially undifferentiated pleomorphic sarcoma, leiomyosarcoma, translocation induced sarcomas (Ewing, synovial, alveolar rhabdomyosarcoma), de-differentiated liposarcoma and osteosarcoma. Lower SUVmax values were observed in sarcomas of low histological grade (grade I), and in rare subtypes of intermediate grade soft tissue sarcoma (e.g. alveolar soft part sarcoma and solitary fibrous tumour). SUVmax variation was noted in malignant peripheral nerve sheath tumours, compared to the histologically benign plexiform neurofibroma, whereas PET-CT could clearly differentiate low from high-grade chondrosarcoma. We identified added utility of PET-CT in addition to MRI and CT in high-grade sarcoma of bone and soft tissues. An estimated 21% overall potential benefit was observed for PET-CT over CT/MRI, and in particular, in 'upstaging' of high-grade disease (from M0 to M1) where an additional 12% of cases were deemed M1 following PET-CT. CONCLUSIONS: PET-CT in high-grade bone and soft tissue sarcoma can add significant benefit to routine CT/MRI staging. Further prospective and multi-centre evaluation of PET-CT is warranted to determine the actual predictive value and cost-effectiveness of PET-CT in directing clinical management of clinically complex and heterogeneous high-grade sarcomas.

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues.

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.

Epithelial-mesenchymal transition and nuclear ß-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent.

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to ß-catenin. Here, we evaluate whether E-cadherin binding can inhibit ß-catenin when there is loss of Adenomatous polyposis coli (APC) from the ß-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear ß-catenin localization, suggesting that E-cadherin inhibits ß-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear ß-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and ß-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits ß-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.

Durable Response of Spinal Chordoma to Combined Inhibition of IGF-1R and EGFR.

Chordomas are rare primary malignant bone tumors arising from embryonal notochord remnants of the axial skeleton. Chordomas commonly recur following surgery and radiotherapy, and there is no effective systemic therapy. Previous studies implicated receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF-1R), in chordoma biology. We report an adult female patient who presented in 2003 with spinal chordoma, treated with surgery and radiotherapy. She underwent further surgery for recurrent chordoma in 2008, with subsequent progression in pelvic deposits. In June 2009, she was recruited onto the Phase I OSI-906-103 trial of EGFR inhibitor erlotinib with linsitinib, a novel inhibitor of IGF-1R/insulin receptor (INSR). Treatment with 100 mg QD erlotinib and 50 mg QD linsitinib was well-tolerated, and after 18 months a partial response was achieved by RECIST criteria. From 43 months, a protocol modification allowed intra-patient linsitinib dose escalation to 50 mg BID. The patient remained stable on trial treatment for a total of 5 years, discontinuing treatment in August 2014. She subsequently experienced further disease progression for which she underwent pelvic surgery in April 2015. Analysis of DNA extracted from 2008 (pre-trial) tissue showed that the tumor harbored wild-type EGFR, and a PIK3CA mutation was detected in plasma, but not tumor DNA. The 2015 (post-trial) tumor harbored a mutation of uncertain significance in ATM, with no detectable mutations in other components of a 50 gene panel, including EGFR, PIK3CA, and TP53. By immunohistochemistry, the tumor was positive for brachyury, the molecular hallmark of chordoma, and showed weak-moderate membrane and cytoplasmic EGFR. IGF-1R was detected in the plasma membrane and cytoplasm and was expressed more strongly in recurrent tumor than the primary. We also noted heterogeneous nuclear IGF-1R, which has been linked with sensitivity to IGF-1R inhibition. Similar variation in IGF-1R expression and subcellular localization was noted in 15 further cases of chordoma. In summary, this exceptionally durable response suggests that there may be merit in evaluating combined IGF-1R/INSR and EGFR inhibition in patients with chordomas that recur following failure of local treatment.

Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics.

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n = 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Quantification of the heterogeneity of prognostic cellular biomarkers in ewing sarcoma using automated image and random survival forest analysis.

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 104 features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.

Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes.

Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2(fl/fl) attenuated the rapid tumour onset promoted by homozygous deletion of p53(fl/fl) . Accelerated carcinoma and sarcoma tumour formation in p53(+/-) females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.

Circulating levels of insulin-like growth factor-II/mannose-6-phosphate receptor in obesity and type 2 diabetes.

OBJECTIVE: The extracellular domain of the insulin-like growth factor II/mannose-6-phosphate receptor (IGF-II/M6P-R) is present in the circulation, but its relationship with plasma IGF-II is largely unknown. As IGF-II appears to be nutritionally regulated, we studied the impact of obesity, type 2 diabetes (T2D) and weight loss on circulating levels of IGF-II and its soluble receptor. METHODS: Twenty-three morbidly obese non-diabetic subjects were studied before and after gastric banding (GB), reducing their BMI from 59.3+/-1.8 to 52.7+/-1.6 kg/m(2). Lean controls (n=10, BMI 24.2+/-0.5 kg/m(2)), moderately obese controls (n=21, BMI 31.8+/-1.0 kg/m(2)) and obese T2D patients (n=20, BMI 32.3+/-0.8 kg/m(2)) were studied before and after a hyperinsulinaemic euglycaemic clamp. RESULTS: Morbidly obese subjects had elevated IGF-II/M6P-R and IGF-II levels, which both decreased following GB (IGF-II/M6P-R: from 0.97+/-0.038 to 0.87+/-0.030 nmol/l, P=0.001; IGF-II: from 134+/-7 to 125+/-6 nmol/l, P=0.01), as did fasting plasma glucose and insulin (P<0.05). However, the metabolic parameters correlated with neither IGF-II nor IGF-II/M6P-R. Obese diabetics had increased IGF-II/M6P-R as compared with lean and obese controls (0.82+/-0.031 vs. 0.70+/-0.033 vs. 0.74+/-0.026 nmol/l; P<0.03) and levels were unaffected by clamp. In the latter cohort, IGF-II/M6P-R but not IGF-II correlated with HbA1c, and fasting plasma C-peptide, insulin and glucose (0.34