Survey of public attitudes towards imminent death donation in the United States.

Imminent death donation (IDD) is described as living organ donation prior to a planned withdrawal of life-sustaining care in an imminently dying patient. Although IDD was ethically justified by United Network for Organ Sharing, the concept remains controversial due to presumed lack of public support. The aim of this study was to evaluate the public's attitudes towards IDD. A cross-sectional survey was conducted of US adults age >18 years (n = 2644). The survey included a case scenario of a patient with a devastating brain injury. Responses were assessed on a 5-point Likert scale. Results showed that 68% - 74% of participants agreed or strongly agreed with IDD when posed as a general question and in relation to the case scenario. Participants were concerned about "recovery after a devastating brain injury" (34%), and that "doctors would not try as hard to save a patient's life" (33%). Only 9% of participants would be less likely to trust the organ donation process. In conclusion, our study demonstrates strong public support for IDD in the case of a patient with a devastating brain injury. Notably, participants were not largely concerned with losing trust in the organ donation process. These results justify policy change towards imminent death donation.

25-hydroxyvitamin D serum levels in patients with high risk resected melanoma treated in an adjuvant bevacizumab trial.

BACKGROUND: Studies evaluating a relationship of vitamin D in patients with primary melanoma have consistently identified an inverse correlation with Breslow thickness, but an inconsistent impact on survival. Vitamin D in later stages of melanoma has been less studied. METHODS: Vitamin D was measured in serum from 341 patients with resected stage IIB-IIIC melanoma recruited to the AVAST-M adjuvant melanoma randomised trial, collected prior to randomisation, then at 3 and 12 months. Vitamin D levels were compared with patient demographics, known melanoma prognostic factors, disease-free interval (DFI) and overall survival (OS). RESULTS: A total of 73% patients had stage III melanoma, 32% were enroled (and therefore tested) >1 year after primary melanoma diagnosis. Median pre-randomisation vitamin D level was 56.5 (range 12.6-189.0 nmol/L). Vitamin D levels did not significantly vary over 12 months (p = 0.24). Individual pre-randomisation vitamin D levels did not differ significantly for Breslow thickness, tumour ulceration, or disease stage. Neither did pre-randomisation vitamin D predict for DFI (HR = 0.98 per 10 nmol/L increase; 95% confidence interval (CI) 0.93-1.04, p = 0.59) or OS (HR = 0.96 per 10 nmol/L increase, 95% CI 0.90-1.03, p = 0.31). For stage II patients, DFI improved with higher pre-randomisation vitamin D levels for those on bevacizumab (HR = 0.74 per 10 nmol nmol/L increase; 95% CI 0.56-0.97), but not for the observation arm (HR = 1.07 per 10 nmol/L increase; 95% CI 0.85-1.34). CONCLUSIONS: In this stage II/III melanoma cohort, vitamin D did not correlate with known prognostic markers, nor predict for DFI or OS, but there was some evidence of benefit for patients with stage II disease treated with bevacizumab.

MRE11 as a Predictive Biomarker of Outcome After Radiation Therapy in Bladder Cancer.

PURPOSE: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials. METHODS AND MATERIALS: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom. RESULTS: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival. CONCLUSIONS: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes.

Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation.

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy.

Vaccination of colorectal cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) induces immune responses which correlate with disease control: a phase I/II trial.

PURPOSE: The highly attenuated strain of vaccinia virus, modified vaccinia Ankara (MVA), encoding the tumor antigen 5T4 (termed TroVax), has been evaluated in an open-label phase I/II study in colorectal cancer patients. The primary objectives were to assess the safety and immunogenicity of ascending doses of TroVax and to determine the biodistribution of the vector. EXPERIMENTAL DESIGN: TroVax was given to 22 patients with metastatic colorectal cancer. Seventeen patients received doses of TroVax ranging from 5 x 10(7) up to 5 x 10(8) plaque-forming units at 0, 4, and 8 weeks and were considered to be evaluable for assessment of immunologic responses. Both antibody and cellular responses specific for the tumor antigen 5T4 and the viral vector were monitored throughout the study. RESULTS: TroVax was well tolerated in all patients with no serious adverse events attributed to vaccination. Of 17 evaluable patients, 16 showed 5T4-specific cellular responses whereas 14 had detectable antibody levels following vaccination. TroVax was able to boost 5T4-specific immune responses in the presence of MVA neutralizing antibodies. Periods of disease stabilization ranging from 3 to 18 months were observed in five patients, all of whom mounted 5T4-specific immune responses. Furthermore, statistical analysis showed a positive association between the development of a 5T4 (but not MVA) antibody response and patient survival or time to disease progression. CONCLUSION: These data indicate that vaccination with TroVax is safe and well tolerated and that immune responses to 5T4 can be induced without any evidence of autoimmune toxicity. Furthermore, 5T4-specific antibody responses correlate with evidence of disease control.

Salt-Inducible Kinase 2 Couples Ovarian Cancer Cell Metabolism with Survival at the Adipocyte-Rich Metastatic Niche.

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer.

Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies.

Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p<2(-16)), which was not found in patients without cancer (n=108). Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n=100), and common in BRCA1-BRCA2 mutation carriers (n=71) who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.

Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy.

Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific scFv fusion proteins.

Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers.

Active treatment of murine tumors with a highly attenuated vaccinia virus expressing the tumor associated antigen 5T4 (TroVax) is CD4+ T cell dependent and antibody mediated.

5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26-h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8+ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4+ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4+ and CD8+ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26-h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4+ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model.

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.