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Scope Micronutrients are in small amounts in foods, act in concert, and require variable amounts of time to see changes in health and risk for disease. These first principles are incorporated into an intervention study designed to develop new experimental strategies for setting target recommendations for food bioactives for populations and individuals. Methods and results A 6-week multivitamin/mineral intervention is conducted in 9-13 year olds. Participants (136) are (i) their own control (n-of-1); (ii) monitored for compliance; (iii) measured for 36 circulating vitamin forms, 30 clinical, anthropometric, and food intake parameters at baseline, post intervention, and following a 6-week washout; and (iv) had their ancestry accounted for as modifier of vitamin baseline or response. The same intervention is repeated the following year (135 participants). Most vitamins respond positively and many clinical parameters change in directions consistent with improved metabolic health to the intervention. Baseline levels of any metabolite predict its own response to the intervention. Elastic net penalized regression models are identified, and significantly predict response to intervention on the basis of multiple vitamin/clinical baseline measures. Conclusions The study design, computational methods, and results are a step toward developing recommendations for optimizing vitamin levels and health parameters for individuals.

The B vitamins folic acid, vitamin B12 and B6 are essential for neuronal function, and severe deficiencies have been linked to increased risk of neurodevelopmental disorders, psychiatric disease and dementia. Polymorphisms of genes involved in B vitamin absorption, metabolism and function, such as methylene tetrahydrofolate reductase (MTHFR), cystathionine β synthase (CβS), transcobalamin 2 receptor (TCN2) and methionine synthase reductase (MTRR), have also been linked to increased incidence of psychiatric and cognitive disorders. However, the effects of these polymorphisms are often quite small and many studies failed to show any meaningful or consistent associations. This review discusses previous findings from clinical studies and highlights gaps in knowledge. Future studies assessing B vitamin-associated polymorphisms must take into account not just traditional demographics, but subjects’ overall diet, relevant biomarkers of nutritional status and also analyze related genetic factors that may exacerbate behavioral effects or nutritional status.

The limited therapeutic options available for patients with metastatic papillary thyroid carcinomas (PTC) and follicular thyroid carcinomas (FTC) necessitates the development of novel therapies. Identification of somatic rearrangements of the tyrosine kinase domain of the RET gene in PTC have improved our understanding of thyroid tumorigenesis. Sunitinib is active against the RET kinase and has both antineoplastic and antiangiogenic properties. Its role in the treatment of patients with thyroid carcinoma has yet to be evaluated in clinical trials. Two patients with progressive metastatic thyroid carcinoma (case 1: PTC, and case 2: FTC) were enroled in a phase I clinical trial to evaluate positron emission tomography (PET) in the monitoring of response to sunitinib. Tumour biopsies and PET were performed at baseline and 4 weeks after the commencement of sunitinib. Activation of the RET kinase pathway was evaluated using immunohistochemistry (IHC) and western blot analysis of total phosphorylated tyrosine and downstream signalling targets of the RET pathway. Both patients demonstrated sustained clinical responses to sunitinib over a duration of 4 years. In case 1, (PTC) PET confirmed evidence of a partial metabolic response, and IHC and western blot analysis demonstrated inhibition of the RET kinase pathway posttreatment. In case 2, (FTC) PET confirmed stable disease after sunitinib. IHC staining of the tumour showed low total phosphorylated tyrosine staining at baseline which did not change after treatment. These case studies highlight potential activity of sunitinib in patients with metastatic thyroid carcinoma. Sunitinib seems to be a promising agent in the treatment of thyroid cancers and this requires validation in future clinical trials.

In vivo models that recapitulate oncogene-dependent tumorigenesis will greatly facilitate development of molecularly targeted anticancer therapies. We have developed a model based on activating mutations in c-KIT in gastrointestinal stromal tumors (GISTs). This model comprises murine tumors of FDC-P1 cell lines expressing c-KIT mutations that render the tumors either responsive (V560G) or resistant (D816V) to the small-molecule c-KIT inhibitor, imatinib. Clinically, GIST response to imatinib is associated with rapid reduction in fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET), preceding changes in conventional response criteria by several weeks. Using the FDC-P1 model in small animal PET, FDG uptake into tumors expressing the c-KIT V560G mutation was significantly reduced as early as 4 hours after imatinib treatment. In contrast, no change in FDG uptake was observed in resistant c-KIT D816V-expressing tumors after 48 hours of imatinib treatment. Consistent with the PET results, expression of the glucose transporter, GLUT1, was significantly reduced in V560G tumors at 4 hours, preceding changes in markers of proliferation by several hours. In vitro, imatinib treatment of V560G cells resulted in a reduction of glucose transporter numbers at the cell surface and decreased glucose uptake well before changes in cell viability. Notably, decreased ambient glucose concentrations enhanced the cytotoxic effect of imatinib. Taken together, these data account for the rapidity and significance of the PET response to imatinib and suggest that metabolic effects may contribute to imatinib cytotoxicity. Further, the FDC-P1 model represents a very useful paradigm for molecularly targeted drug development.

Understanding the regulation of Akt has been of major interest for elucidating the control of normal cellular physiology as well as malignant transformation. The paradigm for activation of Akt involves phosphatidylinositol 3-kinase-dependent membrane localization followed by activating phosphorylation of Thr-308 and Ser-473. Many of the activating signals for Akt involve the stimulation of receptor and non-receptor tyrosine kinases, and the most potent activator known is the tyrosine phosphatase inhibitor pervanadate, highlighting a possible role for tyrosine phosphorylation in the regulation of the enzyme. In this study we show that activation of Akt by pervanadate or serum is associated with tyrosine phosphorylation of Akt. In addition, in SKOV3 ovarian carcinoma cells that exhibit high basal levels of Akt activity, Akt was tyrosine-phosphorylated in the basal state, and this phosphorylation was further enhanced by both pervanadate and insulin-like growth factor-1. We have used NH(2)-terminal sequencing and phosphate release analysis to directly identify Tyr-474 as the site of tyrosine phosphorylation. Substitution of Tyr-474 with phenylalanine abolished tyrosine phosphorylation of Akt and resulted in up to 55% inhibition of Akt activation, indicating phosphorylation at Tyr-474 is required for full activation of the kinase. Our data identifies a novel regulatory mechanism for this pleiotropic enzyme that may be applicable to the AGC family of protein kinases given the conserved nature of the COOH-terminal hydrophobic motif containing Tyr-474.

Summary Background Locally advanced skin cancers including squamous cell carcinoma (SCC) of the skin are increasing in incidence. Patients are often elderly with significant comorbidities and therapy can be difficult. New targeted therapies, such as treatment directed at the epidermal growth factor receptor (EGFR), may be effective and less toxic in these patients. However, before designing appropriate clinical trials it is necessary to characterize the expression and activation of targets such as the EGFR to evaluate the rationale of using EGFR inhibitors (EGFRIs) in the treatment of this type of cancer. Objectives To characterize the expression and activation by phosphorylation of EGFR in SCC of the skin by quantitative Western blotting using the LiCor immunofluorescence detection system with validation by immunohistochemistry. Secondary objectives were to evaluate downstream targets of EGFR expression and activation in SCC of the skin and to examine the associations between EGFR, pathological features and clinical behaviour of these tumours. Methods Twenty-one mainly locally advanced skin SCCs collected in our institution and stored in our tissue bank over a 4-year period were used for the study. Results Nine of 21 (43%) tumours expressed EGFR above background. Of those nine, five expressed phosphorylated EGFR. There was no correlation with downstream activation of canonical signalling pathways, pathological features or clinical behaviour. Conclusions EGFR is expressed in a minority of tumours and then is not always activated. These results show that, before designing a trial with a targeted agent such as an EGFRI in SCC of the skin, it is important to verify the presence of the appropriate target to maximize the best outcome.

This study was designed as "proof of concept" for a drug development model utilising multi-tracer serial small animal PET imaging to characterise tumour responses to molecularly targeted therapy.Mice bearing subcutaneous A431 human squamous carcinoma xenografts (n=6-8) were treated with the pan-Erb-B inhibitor CI-1033 or vehicle and imaged serially (days 0, 3 and 6 or 7) with [(18)F]fluorodeoxyglucose, [(18)F]fluoro-L: -thymidine, [(18)F]fluoro-azoazomycinarabinoside or [(18)F]fluoromisonidazole. Separate cohorts (n=3) were treated identically and tumours were assessed ex vivo for markers of glucose metabolism, proliferation and hypoxia.During the study period, mean uptake of all PET tracers generally increased for control tumours compared to baseline. In contrast, tracer uptake into CI-1033-treated tumours decreased by 20-60% during treatment. Expression of the glucose transporter Glut-1 and cell cycle markers was unchanged or increased in control tumours and generally decreased with CI-1033 treatment, compared to baseline. Thymidine kinase activity was reduced in all tumours compared to baseline at day 3 but was sevenfold higher in control versus CI-1033-treated tumours by day 6 of treatment. Uptake of the hypoxia marker pimonidazole was stable in control tumours but was severely reduced following 7 days of CI-1033 treatment.CI-1033 treatment significantly affects tumour metabolism, proliferation and hypoxia as determined by PET. The PET findings correlated well with ex vivo biomarkers for each of the cellular processes studied. These results confirm the utility of small animal PET for evaluation of the effectiveness of molecularly targeted therapies and simultaneously definition of specific cellular processes involved in the therapeutic response.