Wnt/β-catenin signaling plays key roles in several developmental and pathological processes. Domains of Wnt expression have been extensively investigated in the mouse, but the tissues receiving the signal remain largely unidentified. To define which cells respond to activated β-catenin during mammalian development, we generated the β-catenin-activated transgene driving expression of nuclear β-galactosidase reporter (BAT-gal) transgenic mice, expressing the lacZ gene under the control of β-catenin/T cell factor responsive elements. Reporter gene activity is found in known organizing centers, such as the midhindbrain border and the limb apical ectodermal ridge. Moreover, BAT-gal expression identifies novel sites of Wnt signaling, like notochord, endothelia, and areas of the adult brain, revealing an unsuspected dynamic pattern of β-catenin transcriptional activity. Expression of the transgene was analyzed in mutant backgrounds. In lipoprotein receptor-related protein 6-null homozygous mice, which lack a Wnt coreceptor, BAT-gal staining is absent in mutant tissues, indicating that BAT-gal mice are bona fide in vivo indicators of Wnt/β-catenin signaling. Analyses of BAT-gal expression in the adenomatous polyposis coli (multiple intestinal neoplasia/+) background revealed βcatenin transcriptional activity in intestinal adenomas but surprisingly not in normal crypt cells. In summary, BAT-gal mice unveil the entire complexity of Wnt/β-catenin signaling in mammals and have broad application potentials for the identification of Wnt-responsive cell populations in development and disease.

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.

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF₁₆₅ and antiangiogenic VEGF₁₆₅b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF_xxxb, but there was a variable upregulation of VEGF_xxx and downregulation of VEGF_xxxb in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF_xxxb, whereas colonic carcinoma cells expressed predominantly VEGF_xxx. However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF_xxxb to predominantly VEGF_xxx. VEGF₁₆₅b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF₁₆₅b bound to bevacizumab with similar affinity as VEGF₁₆₅. However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF₁₆₅, it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF₁₆₅b. Both bevacizumab and anti-VEGF₁₆₅b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF₁₆₅b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.

Pancreatic cancer is the fifth most common cause of cancer death. Identification of defined patient groups based on a prognostic index may improve the prediction of survival and selection of therapy. Many prognostic factors have been identified often based on retrospective, underpowered studies with unclear analyses. Data from 653 patients were analysed. Continuous variables are often simplified assuming a linear relationship with log hazard or introducing a step function (dichotomising). Misspecification may lead to inappropriate conclusions but has not been previously investigated in pancreatic cancer studies. Models based on standard assumptions were compared with a novel approach using nonlinear fractional polynomial (FP) transformations. The model based on FP-transformed covariates was most appropriate and confirmed five previously reported prognostic factors: albumin, CA19-9, alkaline phosphatase, LDH and metastases, and identified three additional factors not previously reported: WBC, AST and BUN. The effects of CA19-9, alkaline phosphatase, AST and BUN may go unrecognised due to simplistic assumptions made in statistical modelling. We advocate a multivariable approach that uses information contained within continuous variables appropriately. The functional form of the relationship between continuous covariates and survival should always be assessed. Our model should aid individual patient risk stratification and the design and analysis of future trials in pancreatic cancer.

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. 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. 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. 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.