Charles J. Link

Borderline ovarian tumors

Borderline tumor of the ovary (BOT) is an epithelial tumor with a low rate of growth and a low potential to invade or metastasize. This tumor often is associated with a significantly better prognosis than epithelial ovarian cancer. Most tumors are either serous or mucinous in histology and present as early stage lesions. However, stage III lesions with peritoneal implants are not uncommon. Patients with early stage lesions have an excellent prognosis. Patients with higher stage lesions have a worse prognosis. Long-term follow-up of patients with BOT is required since the tumor can recur up to 20 years after the initial diagnosis. Recently, investigators have begun to identify subsets of patients with a worse prognosis, such as patients with aneuploid tumors. Treatment for early stage lesions is surgical and conservative surgery can be accomplished successfully in younger patients who desire to maintain fertility. Treatment for later stage lesions has been approached in a variety of ways. All approaches initially begin with maximal cytoreductive surgery. Studies suggest that early stage disease should be managed with surgery alone. Conflicting results on the usefulness of adjuvant therapy for patients with later stage disease have been obtained. At this time, the usefulness of adjuvant therapy for advanced disease remains undetermined. Further understanding of the basis of the disease and analysis of specific higher risk subsets might identify patients in whom adjuvant therapy could be tested in the setting of controlled clinical trials.

A phase I study of indoximod in patients with advanced malignancies

Purpose Indoximod is an oral inhibitor of the indoleamine 2,3-dioxygenase pathway, which causes tumor-mediated immunosuppression. Primary endpoints were maximum tolerated dose (MTD) and toxicity for indoximod in patients with advanced solid tumors. Secondary endpoints included response rates, pharmacokinetics, and immune correlates. Experimental Design Our 3+3 phase I trial comprised 10 dose levels (200, 300, 400, 600, and 800 mg once/day; 600, 800, 1200, 1600, and 2000 mg twice/day). Inclusion criteria were measurable metastatic solid malignancy, age ≥18 years, and adequate organ/marrow function. Exclusion criteria were chemotherapy ≤ 3 weeks prior, untreated brain metastases, autoimmune disease, or malabsorption. Results In 48 patients, MTD was not reached at 2000 mg twice/day. At 200 mg once/day, 3 patients previously treated with checkpoint inhibitors developed hypophysitis. Five patients showed stable disease >6 months. Indoximod plasma AUC and Cmax plateaued above 1200mg. Cmax (∼12 μM at 2000 mg twice/day) occurred at 2.9 hours, and half-life was 10.5 hours. C reactive protein (CRP) levels increased across multiple dose levels. Conclusions Indoximod was safe at doses up to 2000 mg orally twice/day. Best response was stable disease >6 months in 5 patients. Induction of hypophysitis, increased tumor antigen autoantibodies and CRP levels were observed.

Adenovirus-p53-mediated gene therapy of anaplastic large cell lymphoma with t(2;5) in a nude mouse model

Adenovirus-p53-mediated apoptosis has been extensively evaluated in animal xenografts derived from human epithelial tumors and recently began testing in phase I clinical trials, but has not been evaluated for lymphoid malignancies. Cell lines derived from anaplastic large cell lymphoma (ALCL) carrying the t(2;5) translocation are efficiently transduced by adenoviral vector expressing p53 and undergo apoptosis. To test the in vivo efficiency of adenovirus-mediated-p53 expression and apoptosis induction, SUDHL-1 cells (derived from human ALCL) were injected subcutaneously into athymic nude mice. Cells from the xenograft had typical morphology of human ALCL by standard hematoxylin-eosin staining, CD5+, CD45+ and CD30+ immunophenotype, the t(2;5) translocation by PCR. Six tumors from an initial set of mice were evaluated for apoptosis by TUNEL and for necrosis by hematoxylin-eosin staining 48–72 h after injection with 1 × 108 p.f.u. of AdWTp53 (adenoviral vector expressing p53), of AdNull (adenoviral vector backbone) and PBS (mock), respectively. TUNEL staining was positive only in tumors injected with AdWTp53 and was mainly localized around the needle track. Differences of the means of the counts of the necrotic cells were statistically significant at P = 0.02 between AdWTp53 and mock and only borderline between AdWTp53 and AdNull. Twenty-three tumors from a separate set of mice were subsequently injected with AdWTp53, AdNull and PBS and evaluated for in vivo tumor response. Three total injections of viral vectors (1 × 108 p.f.u.) and PBS were given every 48–72 h. Only tumors injected with AdWTp53 showed tumor growth inhibition with a mean final tumor volume that was statistically significantly smaller than AdNull (P = 0.007) and mock (P = 0.002). Based on these results we foresee a potential application of adenovirus-mediated p53 apoptosis as gene therapy of lymphomas.

Desferrioxamine treatment increases the genomic stability of Ataxia-telangiectasia cells

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl(2) in the colony forming-efficiency assay and fail to demonstrate a FeCl(2)-induced G(2) checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.

A novel herpesvirus amplicon system for in vivo gene delivery.

For gene therapy approaches to succeed, improved vector systems are needed that combine a large carrying capacity with high transduction efficiency in vivo. Towards this goal, we have developed a novel herpes simplex virus (HSV) amplicon vector, pHE, which contains an HSV-1 replication origin (ori S) and packaging sequence that permit vector replication and packaging into HSV-1 capsids. The vector also contains the Epstein–Barr virus (EBV) unique latent replication origin (ori P) sequence and a modified EBNA-1 gene to allow the vector to be maintained as an episome in transfected E5 helper cells. This system allows for efficient packaging of high-titer vector since the E5 cells are first selected for the presence of the pHE vector before helper virus infection. The infectious pHE vector has efficient transgene expression in a variety of human cell lines in vitro. Stereotactic injection of pHE vector supernatant into the rat brain resulted in high, localized reporter gene expression. Finally, the pHE vector could carry a stable 21 kb DNA payload into HSV virions. This pHE vector system should have a broad range of gene transfer applications.

Safety and immunogenicity of the rVSV∆G-ZEBOV-GP Ebola virus vaccine candidate in healthy adults: a phase 1b randomised, multicentre, double-blind, placebo-controlled, dose-response study

BACKGROUND The 2014 Zaire Ebola virus outbreak highlighted the need for a safe, effective vaccine with a rapid onset of protection. We report the safety and immunogenicity of the recombinant vesicular stomatitis virus-Zaire Ebola virus envelope glycoprotein vaccine (rVSV∆G-ZEBOV-GP) across a 6 log10 dose range in two sequential cohorts. METHODS In this phase 1b double-blind, placebo-controlled, dose-response study we enrolled and randomly assigned healthy adults (aged 18-61 years) at eight study sites in the USA to receive a single injection of vaccine or placebo, administered by intramuscular injection. In cohort 1, participants were assigned to receive 3 × 103, 3 × 104, 3 × 105, or 3 × 106 PFU doses of rVSV∆G-ZEBOV-GP or placebo. In cohort 2, participants were assigned to receive 3 × 106, 9 × 106, 2 × 107, or 1 × 108 PFU doses of rVSV∆G-ZEBOV-GP or placebo. Participants were centrally allocated by the study statistician to vaccine groups or placebo through computer-generated randomisation lists. The primary safety outcome was incidence of adverse events within 14 days in the modified intention-to-treat population (all randomly assigned participants who received vaccine or placebo), and the primary outcome for immunogenicity was IgG ELISA antibody titres at day 28 in the per-protocol population. Surveillance was enhanced for arthritis and dermatitis through to day 56. This study is registered with ClinicalTrials.gov, number NCT02314923. FINDINGS Between Dec 26, 2014, and June 8, 2015, 513 participants were enrolled and randomly assigned; one was not immunised because of unsuccessful phlebotomy. In cohort 1, 256 participants received vaccine (3 × 103 [n=64], 3 × 104 [n=64], 3 × 105 [n=64], or 3 × 106 PFU [n=64]) and 74 received placebo. In cohort 2, 162 participants received vaccine (3 × 106 [n=20], 9 × 106 [n=47], 2 × 107 [n=47], or 1 × 108 PFU [n=48]) and 20 received placebo. Most adverse events occurred in the first day after vaccination, and were mild to moderate in intensity, of a short duration, and more frequent at high vaccine doses (9 × 106 PFU and greater). At the 2 × 107 PFU dose (used in phase 3 trials), the most common local adverse events versus placebo within the first 14 days were arm pain (57·4% [27 of 47] vs 7·4% [seven of 94]) and local tenderness (59·6% [28 of 47] vs 8·5% [eight of 94]). The most common systemic adverse events at the 2 × 107 PFU dose versus placebo, occurring in the first 14 days, were headache (46·8% [22 of 47] vs 27·7% [26 of 94]), fatigue (38·3% [18 of 47] vs 19·1% [18 of 94]), myalgia (34·0% [16 of 47] vs 10·6% [10 of 94]), subjective fever (29·8% [14 of 47] vs 2·1% [two of 94]), shivering or chills (27·7% [13 of 47] vs 7·4% [seven of 94]), sweats (23·4% [11 of 47] vs 3·2% [three of 94]), joint aches and pain (19·1% [nine of 47] vs 7·4% [seven of 94]), objective fever (14·9% [seven of 47] vs 1·1% [one of 94]), and joint tenderness or swelling (14·9% [seven of 47] vs 2·1% [two of 94]). Self-limited, post-vaccination arthritis occurred in 4·5% (19 of 418) of vaccinees (median onset 12·0 days [IQR 10-14]; median duration 8·0 days [6-15]) versus 3·2% (three of 94) of controls (median onset 15·0 days [6-20]; median duration 47·0 days [37-339]), with no apparent dose relationship. Post-vaccination dermatitis occurred in 5·7% (24 of 418) of vaccinees (median onset 9·0 days [IQR 2-12]; median duration 7·0 days [4-9]) versus 3·2% (three of 94) of controls (median onset 5·0 days [3-53]; median duration 33·0 days [5-370]). A low-level, transient, dose-dependent viraemia occurred in concert with early reactogenicity. Antibody responses were observed in most participants by day 14. IgG and neutralising antibody titres were dose-related (p=0·0003 for IgG ELISA and p<0·0001 for the 60% plaque-reduction neutralisation test [PRNT60] by linear trend). On day 28 at the 2 × 107 PFU dose, the geometric mean IgG ELISA endpoint titre was 1624 (95% CI 1146-2302) and seroconversion was 95·7% (95% CI 85·5-98·8); the geometric mean neutralising antibody titre by PRNT60 was 250 (176-355) and seroconversion was 95·7% (85·5-98·8). These robust immunological responses were sustained for 1 year. INTERPRETATION rVSV∆G-ZEBOV-GP was well tolerated and stimulated a rapid onset of binding and neutralising antibodies, which were maintained through to day 360. The immunogenicity results support selection of the 2 × 107 PFU dose. FUNDING Biomedical Advanced Research and Development Authority, US Department of Health and Human Services.

Rat sodium iodide symporter allows using lower dose of 131I for cancer therapy

Efficient gene delivery is a critical obstacle for gene therapy that must be overcome. Until current limits of gene delivery technology are solved, identification of systems with bystander effects is highly desirable. As an anticancer agent, radioactive iodine 131I has minimal toxicity. The physical characteristics of 131I decay allow radiation penetration within a local area causing bystander killing of adjacent cells. Accumulation of 131I mediated by the sodium iodide symporter (NIS) provides a highly effective treatment for well-differentiated thyroid carcinoma. Other types of cancer could also be treated by NIS-mediated concentration of lethal 131I radiation in tumor cells. Our group and others previously reported that a significant antitumor effect in mice was achieved after adenoviral delivery of rat or human NIS gene following administration of 3 mCi of 131I. We have also demonstrated 5–6-fold greater uptake of 125I by rat NIS over human NIS in human cancer cells. Recently, we reported the capability of the rat NIS and 131I to effectively induce growth arrest of relatively large tumors (approximately 800 mm3) in an animal model. In the present work tumor growth inhibition was achieved using adenoviral delivery of the rat NIS gene and 1 mCi of 131I (one-third of the dose used in earlier reports). We also demonstrated that a higher concentration of 123I was accumulated in the NIS-expressing tumors than in the thyroid 20 min after radioiodine administration. The highest intratumoral radioiodine concentration was observed along the needle track; however, the rat NIS-131I effectively induced growth arrest of tumor xenografts in mice through its radiological bystander effect. Importantly, the rat NIS allowed reducing the injected radioiodine dose by 70% with the same antitumor efficacy in pre-established tumors. These results suggest that the rat NIS gene may be advantageous compared to the human gene in its ability to enhance intratumoral 131I uptake.

Chapter 29 – HyperAcute Vaccines: A Novel Cancer Immunotherapy

The hyperacute rejection of a xenotransplant is characterized by a complement-antibody mediated immune response dependent on αGal epitopes. Animal studies confirm that αGal epitopes expressed on allogeneic tumor vaccines elicit a potent T-cell-dependent antitumor immunity. Based on these immunologic reactions, we hypothesized that the hyperacute rejection mechanism could be exploited to alter antigen processing resulting in a novel therapeutic approach to treat human malignancies. Clinical trials data confirm that an immediate hypersensitivity response directed toward a vaccine composed of genetically modified allogeneic tumor cells expressing the xenoantigen αGal (HyperAcute vaccines) constitutes a polyvalent tumor cell vaccine with signs of clinical efficacy, concomitant to eliciting both a humoral IgG response as well as T-cell-mediated antitumor immunity. This conceptually innovative immunotherapy degrades tumoral immune escape and portends a promising genetic engineering tactic for the cost-effective development of a generally applicable human cancer vaccine principle with minimal toxicity. Encouraging results support additional clinical immunotherapy studies using HyperAcute vaccines.

Radiometric Characterization Methods For Infrared Countermeasures Systems

Infrared countermeasures systems used to protect aircraft must produce a powerful infrared radiation pattern spread over a wide range of angles, and with a complicated modulation in time. The measurement of this radiation with respect to the parameters of time, angle, and wavelength is described. The radiometer measurement equation is examined to see what approximations are necessary for a solution in the terms desired. The measurement apparatus is described along with how it satisfies the measurement equation approximations. A two-step procedure was required for measurement of the spectral radiant intensity. First, the relative spectral output was determined using a monochromator, and then a scaling factor is determined for converting this to absolute units. An electrically activated shutter was used for the radiometer reference rather than the usual chopper. The detector was used in the DC coupled mode and a digital oscilloscope was used under computer control to acquire data. The results obtained were intended mainly for setting parameters for simulation studies of the effectiveness of these countermeasures systems. The manu-facturers of these systems also have measurement facilities in their own plants. A comparison is made between these different techniques, discussing the different approximations required and the advantages or disadvantages of these different methods.