Genetic studies of GRN and IFT74 in amyotrophic lateral sclerosis

There is increasing evidence of a clinical, neuropathological and genetic overlap between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We conducted a case–control study using a UK dataset to test the hypothesis that polymorphisms in two FTD-related genes (GRN and FT74) are associated with increased susceptibility to ALS. We evaluated the majority of known genetic variability in IFT74 and GRN. The results revealed that the common variations in IFT74 and GRN neither constitute strong ALS risk factors nor modify the age-at-onset. However, the possibility of a modest risk effect remains to be assessed in large datasets.     

Enhanced in vitro and in vivo cytotoxicity of combined reovirus and radiotherapy.

PURPOSE: To test combination treatment schedules of reovirus and radiation in human and murine tumor cells in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro cytotoxicity and cell cycle effects of reovirus given alone and combined with radiotherapy were assessed by colorimetric, tissue culture infectious dose 50, and fluorescence-activated cell sorting-based assays. Interactions between the agents were evaluated using combination index analysis. The effect of different schedules of reovirus and radiotherapy on viral replication and cytotoxicity was tested in vitro and the combination was assessed in three tumor models in vivo. RESULTS: Characterization of reovirus cytotoxicity in a panel of cell lines yielded a range of sensitivities. Combined reovirus and radiotherapy yielded statistically significantly increased cytotoxicity, particularly in cell lines with moderate susceptibility to reovirus alone. The enhanced cytotoxicity of the combination occurred independently of treatment sequence or schedule. Radiation did not affect viral replication and only reduced reoviral cytotoxicity after clinically irrelevant single doses (>50 Gy). Combination index analysis revealed synergy between radiation (3-10 Gy) and reovirus at multiplicities of infection between 0.001 and 1. Combination treatment significantly increased apoptosis in tumor cells relative to either single-agent treatment. In vivo studies using xenograft and syngeneic tumors showed enhanced activity of the combination relative to reovirus or radiation alone (P < 0.001). CONCLUSIONS: Combining reovirus and radiotherapy synergistically enhances cytotoxicity in a variety of tumor cells in vitro and in vivo. These results offer strong support for translational clinical trials of reovirus plus radiotherapy that have been initiated in the clinic.     

Dendritic cell immunotherapy for urological cancers using cryopreserved allogeneic tumour lysate-pulsed cells: a phase I/II study

OBJECTIVE: To assess the feasibility, toxicity and immunogenicity of dendritic cell (DC)-based immunotherapy in patients with advanced urological cancers. PATIENTS AND METHODS: Patients with hormone-refractory prostate cancer (11) and metastatic renal cell carcinoma (five) received 1-3 x 10(6) intradermal allogeneic tumour lystate-pulsed DCs fortnightly for six vaccinations then monthly until disease progression. Intradermal keyhole limpet haemocyanin was injected near the DCs as the adjuvant. DC vaccine was prepared from buffy coats, then lysate-pulsed, cryopreserved in aliquots, and tested for phenotypic expression and activity in an allogeneic mixed lymphocyte reaction before clinical use. RESULTS: There was no evidence of significant toxicity from vaccine or adjuvant. Delayed-type hypersensitivity skin testing and biopsy revealed a cellular infiltrate to intradermal re-challenge to tumour lysate and adjuvant in almost all patients. In addition, there was increased expression of T helper type 1 cytokines, interferon-gamma-expressing T cell by ELISPOT analysis, but also interleukin-10 in a few patients. Vaccination resulted in a reduction in the level of prostate-specific antigen (PSA) in one patient, a reduction in PSA velocity in a further man and an increased PSA doubling time in six. Two of five patients with renal cell carcinoma had stabilization of disease. CONCLUSION: The cryopreservation and repeated administration of DC vaccine was feasible and not toxic. There was evidence of induction of both humoral and cellular immunity to vaccine and adjuvant in most patients. The use of sequential aliquots of identical cryopreserved vaccine will ensure quality control and greatly facilitate future clinical studies in terms of consistency of vaccine administered and the provision of primed DCs for in vitro assessment of response.     

Assessing the role of insulin‐like growth factors and binding proteins in prostate cancer using Mendelian randomization: Genetic variants as instruments for circulating levels

Circulating insulin‐like growth factors (IGFs) and their binding proteins (IGFBPs) are associated with prostate cancer. Using genetic variants as instruments for IGF peptides, we investigated whether these associations are likely to be causal. We identified from the literature 56 single nucleotide polymorphisms (SNPs) in the IGF axis previously associated with biomarker levels (8 from a genome‐wide association study [GWAS] and 48 in reported candidate genes). In ～700 men without prostate cancer and two replication cohorts (N ～ 900 and ～9,000), we examined the properties of these SNPS as instrumental variables (IVs) for IGF‐I, IGF‐II, IGFBP‐2 and IGFBP‐3. Those confirmed as strong IVs were tested for association with prostate cancer risk, low (< 7) vs. high (≥ 7) Gleason grade, localised vs. advanced stage, and mortality, in 22,936 controls and 22,992 cases. IV analysis was used in an attempt to estimate the causal effect of circulating IGF peptides on prostate cancer. Published SNPs in the IGFBP1/IGFBP3 gene region, particularly rs11977526, were strong instruments for IGF‐II and IGFBP‐3, less so for IGF‐I. Rs11977526 was associated with high (vs. low) Gleason grade (OR per IGF‐II/IGFBP‐3 level‐raising allele 1.05; 95% CI: 1.00, 1.10). Using rs11977526 as an IV we estimated the causal effect of a one SD increase in IGF‐II (～265 ng/mL) on risk of high vs. low grade disease as 1.14 (95% CI: 1.00, 1.31). Because of the potential for pleiotropy of the genetic instruments, these findings can only causally implicate the IGF pathway in general, not any one specific biomarker.     

Analysis of European case-control studies suggests that common inherited variation in mitochondrial DNA is not involved in susceptibility to amyotrophic lateral sclerosis

While some cases of familial ALS can be entirely attributed to known inherited variation, the majority (～ 90%) are sporadic, where the cause(s) are not entirely understood. Both genetic and environmental factors may contribute to susceptibility. Mitochondrial damage, a common feature of neurodegenerative disease, is observed in most patients and inherited polymorphism in the mitochondrial genome has been suggested as a contributing factor. We used an economic and efficient method to test whether such involvement is probable. We genotyped 22 mtDNA coding region SNPs and sequenced the mtDNA hypervariable region 1 to determine the position of each mitochondrial genome within the genealogy of mitochondrial haplotypes in samples of ALS patients (n = 700) and controls (n = 462) from two European populations. We compared haplotype and haplogroup distribution in cases and controls drawn from the same populations. No statistical difference was observed between cases and controls at either the haplogroup or haplotype level (p = ≥ 0.2). In conclusion, it is unlikely that common, shared genetic variants in the mitochondrial genome contribute substantially to ALS. Combining the data with other studies will allow meta-analysis to look for variants with modest effect sizes. The sequencing of complete mitochondrial genomes will be required to assess the role of rare mutations.     

The Profile of Tumor Antigens Which Can be Targeted by Immunotherapy Depends Upon the Tumor's Anatomical Site

Previously, we showed that vesicular stomatitis virus (VSV) engineered to express a cDNA library from human melanoma cells (ASMEL, Altered Self Melanoma Epitope Library) was an effective systemic therapy to treat subcutaneous (s.c.) murine B16 melanomas. Here, we show that intravenous treatment with the same ASMEL VSV-cDNA library was an effective treatment for established intra-cranial (i.c.) melanoma brain tumors. The optimal combination of antigens identified from the ASMEL which treated s.c. B16 tumors (VSV-N-RAS+VSV-CYTC-C+VSV-TYRP-1) was ineffective against i.c. B16 brain tumors. In contrast, combination of VSV-expressed antigens—VSV-HIF-2α+VSV-SOX-10+VSV-C-MYC+VSV-TYRP1—from ASMEL which was highly effective against i.c. B16 brain tumors, had no efficacy against the same tumors growing subcutaneously. Correspondingly, i.c. B16 tumors expressed a HIF-2α^Hi, SOX-10^Hi, c-myc^Hi, TYRP1, N-RAS^loCytc^lo antigen profile, which differed significantly from the HIF-2α^lo, SOX-10^lo, c-myc^lo, TYRP1, N-RAS^HiCytc^Hi phenotype of s.c. B16 tumors, and was imposed upon the tumor cells by CD11b⁺ cells within the local brain tumor microenvironment. Combining T-cell costimulation with systemic VSV-cDNA treatment, long-term cures of mice with established i.c. tumors were achieved in about 75% of mice. Our data show that the anatomical location of a tumor profoundly affects the profile of antigens that it expresses.     

Renal-cell carcinoma: tumour markers, T-cell epitopes, and potential for new therapies

Advanced renal-cell carcinoma is a very difficult tumour to treat, and response rates to biological therapies are less than 20%. The identification of various molecular and cellular markers has led to the development of novel therapies. Despite evaluation of their association with histological subtype, immune infiltration, molecular markers of cell proliferation, p53 mutation, and growth-factor-receptor expression, none of these markers has proved better predictive factors than tumour stage and histological grade. The identification of tumour-associated antigens and the specificity of cellular immune responses have led to the development of targeted immunotherapy with monoclonal antibodies, radioimmunotherapy, and T-cell therapies. In this review, we evaluate a range of markers associated with renal-cell carcinoma and new treatment approaches based on tumour-associated antigens and, in particular, T-cell epitopes.