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Purpose: Enzalutamide, a second-generation androgen antagonist, was approved by the U.S. Food and Drug Administration (FDA) for castration-resistant prostate cancer (CRPC) treatment. Immunotherapy has been shown to be a promising strategy for prostate cancer. This study was performed to provide data to support the combination of enzalutamide and immunotherapy for CRPC treatment.Experimental Design: Male C57BL/6 or TRAMP (transgenic adenocarcinoma of the mouse prostate) prostate cancer model mice were exposed to enzalutamide and/or a therapeutic vaccine targeting Twist, an antigen involved in epithelial-to-mesenchymal transition and metastasis. The physiologic and immunologic effects of enzalutamide were characterized. The generation of Twist-specific immunity by Twist-vaccine was assessed. Finally, the combination of enzalutamide and Twist-vaccine to improve TRAMP mice overall survival was evaluated.Results: Enzalutamide mediated immunogenic modulation in TRAMP-C2 cells. In vivo, enzalutamide mediated reduced genitourinary tissue weight, enlargement of the thymus, and increased levels of T-cell excision circles. Because no changes were seen in T-cell function, as determined by CD4+ T-cell proliferation and regulatory T cell (Treg) functional assays, enzalutamide was determined to be immune inert. Enzalutamide did not diminish the ability of Twist-vaccine to generate Twist-specific immunity. Twist was confirmed as a valid tumor antigen in TRAMP mice by immunohistochemistry. The combination of enzalutamide and Twist-vaccine resulted in significantly increased overall survival of TRAMP mice compared with other treatment groups (27.5 vs. 10.3 weeks). Notably, the effectiveness of the combination therapy increased with disease stage, i.e., the greatest survival benefit was seen in mice with advanced-stage prostate tumors.Conclusions: These data support the combination of enzalutamide and immunotherapy as a promising treatment strategy for CRPC. Clin Cancer Res; 19(22); 6205–18. ©2013 AACR.

PDF file - 2461K, Supplemental Table 1: Cellular immune responses to multiple tumor antigens after combination therapy with MDV3100 and Twist-vaccine.

Numerous reports have now demonstrated that the epithelial-to-mesenchymal transition (EMT) process is involved in solid tumor progression, metastasis, and drug resistance. Several transcription factors have been implicated as drivers of EMT and metastatic progression, including Twist. Overexpression of Twist has been shown to be associated with poor prognosis and drug resistance for many carcinomas and other tumor types. The role of Twist in experimental cancer metastases has been principally studied in the 4T1 mammary tumor model, where silencing of Twist in vitro has been shown to greatly reduce in vivo metastatic spread. Transcription factors such as Twist are generally believed to be “undruggable” because of their nuclear location and lack of a specific groove for tight binding of a small molecule inhibitor. An alternative approach to drug therapy targeting transcription factors driving the metastatic process is T-cell–mediated immunotherapy. A therapeutic vaccine platform that has been previously characterized consists of heat-killed recombinant Saccharomyces cerevisiae (yeast) capable of expressing tumor-associated antigen protein. We report here the construction and characterization of a recombinant yeast expressing the entire Twist protein, which is capable of inducing both CD8+ and CD4+ Twist-specific T-cell responses in vivo. Vaccination of mice reduced the size of primary transplanted 4T1 tumors and had an even greater antitumor effect on lung metastases of the same mice, which was dependent on Twist-specific CD8+ T cells. These studies provide the rationale for vaccine-induced T-cell–mediated therapy of transcription factors involved in driving the metastatic process. Cancer Res; 74(7); 1945–57. ©2014 AACR.

PDF file - 358KB, Expression of EMT marker expression in primary tumor and lung metastases.

PDF file - 138KB, Expression of murine Twist protein in S. cerevisiae Yeast.

PDF file - 208KB, Recombinant Yeast-Twist vaccination does not induce toxicity.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24–88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

Undermining is thought to improve wound outcomes; however, randomized controlled data regarding its efficacy are lacking in humans. The objective of this randomized clinical trial was to determine whether undermining low to moderate tension wounds improves scar cosmesis compared to wound closure without undermining. Fifty-four patients, 18 years or older, undergoing primary linear closure of a cutaneous defect with predicted postoperative closure length of ≥ 3 cm on any anatomic site were screened. Four patients were excluded, 50 patients were enrolled, and 48 patients were seen in follow-up. Wounds were divided in half and one side was randomized to receive either no undermining or 2 cm of undermining. The other side received the unselected intervention. Three months, patients and 2 masked observers evaluated each scar using the Patient and Observer Scar Assessment Scale (POSAS). A total of 50 patients [mean (SD) age, 67.6 (11.5) years; 31 (64.6%) male; 48 (100%) white] were enrolled in the study. The mean (SD) sum of the POSAS observer component scores was 12.0 (6.05) for the undermined side and 11.1 (4.68) for the non-undermined side (P = .60). No statistically significant difference was found in the mean (SD) sum of the patient component for the POSAS score between the undermined side [15.9 (9.07)] and the non-undermined side [13.33 (6.20)] at 3 months. For wounds under low to moderate perceived tension, no statistically significant differences in scar outcome or total complications were noted between undermined wound halves and non-undermined halves.Trail Registry: Clinical trials.gov Identifier NCT02289859. https://clinicaltrials.gov/ct2/show/NCT02289859.

In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 ‘A’ strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine (NCT04591717; https://clinicaltrials.gov/ct2/show/NCT04591717), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

Background Layered closure of cutaneous wounds is a commonly used surgical practice. However, there are studies that suggest the additional layer of epidermal sutures might not be necessary. Objective To compare scar outcomes between the single-layer deep-dermal suture technique and the conventional layered suture technique for primary closure of cutaneous wounds. Methods A total of 49 patients were enrolled in a prospective, randomized, evaluator-blinded, split scar study to compare the conventional bilayered closure technique with the single-layer deep-dermal suturing technique for primary closure of wounds. The primary outcome measure was mean sum Patient and Observer Scar Assessment Scale (POSAS) score at 3 and 12 months. Results At the 3-month follow-up, there was a statistically significant difference in the mean total POSAS scores for both the blinded observer and patients, indicating a preference for the side with the standard layered closure. However, at the 12-month follow-up, this difference was lost, with the exception of scar color, which was significantly more noticeable on the wound side closed with only dermal sutures. Limitation Single-center study. Conclusion Three months after surgery, the layered closure technique resulted in a slightly better scar outcome than the single-layered closure containing only dermal sutures. At 12-months' follow-up, this difference diminished, with scars for both sides appearing similar.

Background Wound edge eversion has been hypothesized to improve aesthetic outcomes after cutaneous wound closure. Data supporting this assertion are sparse. Objective We sought to determine if wound eversion, achieved with interrupted subcuticular sutures, improves aesthetic outcome compared with planar closures. Methods We undertook a prospective, randomized, split-scar intervention in patients who underwent cutaneous surgery. Half of the wound was randomized to an everted or planar repair; the other side received the opposite one. At 3- and 6-month follow-up, both the patient and 2 blinded observers evaluated the wound using the Patient Observer Self-Assessment Scale (POSAS). Results The total observer POSAS score for the everted (13.59, 12.26) and planar (12.91, 12.98) sides did not differ significantly at 3 or 6 months, respectively. Similarly, there was not a significant difference in patient assessment between the everted (16.23, 12.84) and planar (15.07, 12.79) sides at 3 or 6 months, respectively. Finally, there was no significant difference between the 2 closure methods in terms of scar height or width at follow-up. Limitations This was a single-center trial, which used a validated but still subjective scar assessment instrument. Conclusion Wound eversion was not significantly associated with improved overall scar assessments by blinded observers or patient assessment.

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Numerous reports have now demonstrated that the epithelial-to-mesenchymal transition (EMT) process is involved in solid tumor progression, metastasis, and drug resistance. Several transcription factors have been implicated as drivers of EMT and metastatic progression, including Twist. Overexpression of Twist has been shown to be associated with poor prognosis and drug resistance for many carcinomas and other tumor types. The role of Twist in experimental cancer metastases has been principally studied in the 4T1 mammary tumor model, where silencing of Twist in vitro has been shown to greatly reduce in vivo metastatic spread. Transcription factors such as Twist are generally believed to be “undruggable” because of their nuclear location and lack of a specific groove for tight binding of a small molecule inhibitor. An alternative approach to drug therapy targeting transcription factors driving the metastatic process is T-cell–mediated immunotherapy. A therapeutic vaccine platform that has been previously characterized consists of heat-killed recombinant Saccharomyces cerevisiae (yeast) capable of expressing tumor-associated antigen protein. We report here the construction and characterization of a recombinant yeast expressing the entire Twist protein, which is capable of inducing both CD8+ and CD4+ Twist-specific T-cell responses in vivo. Vaccination of mice reduced the size of primary transplanted 4T1 tumors and had an even greater antitumor effect on lung metastases of the same mice, which was dependent on Twist-specific CD8+ T cells. These studies provide the rationale for vaccine-induced T-cell–mediated therapy of transcription factors involved in driving the metastatic process. Cancer Res; 74(7); 1945–57. ©2014 AACR.

Purpose: Enzalutamide, a second-generation androgen antagonist, was approved by the U.S. Food and Drug Administration (FDA) for castration-resistant prostate cancer (CRPC) treatment. Immunotherapy has been shown to be a promising strategy for prostate cancer. This study was performed to provide data to support the combination of enzalutamide and immunotherapy for CRPC treatment. Experimental Design: Male C57BL/6 or TRAMP (transgenic adenocarcinoma of the mouse prostate) prostate cancer model mice were exposed to enzalutamide and/or a therapeutic vaccine targeting Twist, an antigen involved in epithelial-to-mesenchymal transition and metastasis. The physiologic and immunologic effects of enzalutamide were characterized. The generation of Twist-specific immunity by Twist-vaccine was assessed. Finally, the combination of enzalutamide and Twist-vaccine to improve TRAMP mice overall survival was evaluated. Results: Enzalutamide mediated immunogenic modulation in TRAMP-C2 cells. In vivo, enzalutamide mediated reduced genitourinary tissue weight, enlargement of the thymus, and increased levels of T-cell excision circles. Because no changes were seen in T-cell function, as determined by CD4+ T-cell proliferation and regulatory T cell (Treg) functional assays, enzalutamide was determined to be immune inert. Enzalutamide did not diminish the ability of Twist-vaccine to generate Twist-specific immunity. Twist was confirmed as a valid tumor antigen in TRAMP mice by immunohistochemistry. The combination of enzalutamide and Twist-vaccine resulted in significantly increased overall survival of TRAMP mice compared with other treatment groups (27.5 vs. 10.3 weeks). Notably, the effectiveness of the combination therapy increased with disease stage, i.e., the greatest survival benefit was seen in mice with advanced-stage prostate tumors. Conclusions: These data support the combination of enzalutamide and immunotherapy as a promising treatment strategy for CRPC. Clin Cancer Res; 19(22); 6205–18. ©2013 AACR.

Copyright 2016 American Medical Association. All rights reserved.IMPORTANCE: The process of Z-plasty scar revision breaks up a linear scar into multiple parts with the purpose of camouflage and improvement of the cosmetic appearance of surgical scars. Although this postulation guides the practices of many reconstructive surgeons, few studies support improved aesthetic outcomes. OBJECTIVE: To compare the perceived cosmetic appearance of linear scars vs zigzag scars by the general public. DESIGN, SETTING, AND PARTICIPANTS: A computer-generated image of a mature scar was designed in linear and zigzag configurations and overlaid on the faces of standardized headshots of 4 white individuals. Twelve sets of images of linear vs zigzag scars were arranged in side-by-side comparisons in an Internet-based national survey. Respondents rated each scar on the 10-point Patient and Observer Scar Assessment Scale, where a lower score indicated likeness with normal skin and a higher score, the worst scar imaginable. Data were collected from May 1 through June 30, 2013, and analyzed from July 31 to September 1, 2013. MAIN OUTCOMES AND MEASURES: Aesthetic rating of scars by the survey respondents. RESULTS: Eight hundred seventy-six participants responded to the survey (24.5% response rate); of these, 810 completed the survey (379 men [46.1%] and 443 women [53.9%]; 148 [18.0%] were 18 to 29 years, 171 [20.8%] were 30 to 44 years, 290 [35.3%] were 45 to 60 years, and 213 [25.9%] were older than 60 years). Significantly lower scores and better perceived cosmetic outcomes were found for linear scars compared with zigzag scars in every assessed group of images on the Patient and Observer Scar Assessment Scale (mean [SD] scores, 2.9 [1.6] vs 4.5 [2.2], respectively; P < .001). CONCLUSIONS AND RELEVANCE: The lay public has a significantly better perception of the appearance of linear scars compared with zigzag scars in 3 facial locations (temple, cheek, and forehead) of white patients in various age groups.

GlobeImmune's Tarmogen(®) immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated killing of target cells expressing viral and cancer antigens in vivo via a CD8(+) CTL-mediated mechanism. Tarmogens are not neutralized by host immune responses and can be administered repeatedly to boost antigen-specific immunity. Production of the vaccines yields stable off-the-shelf products that avoid the need for patient-specific manufacturing found with other immunotherapeutic approaches. Tarmogens for the treatment of chronic hepatitis B and C and various cancers were well tolerated and immunogenic in phase 1 and 2 clinical trials encompassing600 subjects. The platform is being widely utilized in basic vaccine research and the most rapid path to success in these endeavors follows from optimal immunoassay selection and execution. This chapter provides detailed methods for the construction and preclinical immunogenicity testing of yeast-based immunotherapeutic products to support the rapid and efficient use of this versatile technology.

GlobeImmune's Tarmogen(®) immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated killing of target cells expressing viral and cancer antigens in vivo via a CD8(+) CTL-mediated mechanism. Tarmogens are not neutralized by host immune responses and can be administered repeatedly to boost antigen-specific immunity. Production of the vaccines yields stable off-the-shelf products that avoid the need for patient-specific manufacturing found with other immunotherapeutic approaches. Tarmogens for the treatment of chronic hepatitis B and C and various cancers were well tolerated and immunogenic in phase 1 and 2 clinical trials encompassing >600 subjects. The platform is being widely utilized in basic vaccine research and the most rapid path to success in these endeavors follows from optimal immunoassay selection and execution. This chapter provides detailed methods for the construction and preclinical immunogenicity testing of yeast-based immunotherapeutic products to support the rapid and efficient use of this versatile technology.

Most small nucleolar RNAs (snoRNAs) guide rRNA nucleotide modifications, some participate in pre-rRNA cleavages, and a few have both functions. These activities involve direct base-pairing of the snoRNA with pre-rRNA using different domains. It is not known if the modification and processing functions occur independently or in a coordinated manner. We address this question by mutational analysis of a yeast box H/ACA snoRNA that mediates both processing and modification. This snoRNA (snR10) contains canonical 5'- and 3'-hairpin structures with a guide domain for pseudouridylation in the 3' hairpin. Our functional mapping results show that: (i) processing requires the 5' hairpin exclusively, in particular a 7-nt element; (ii) loss of the 3' hairpin or pseudouridine does not affect rRNA processing; (iii) a single nucleotide insertion in the guide domain shifts modification to an adjacent uridine in rRNA, and severely impairs both processing and cell growth; and (iv) the deleterious effects of the insertion mutation depend on the presence of the processing element in the 5' hairpin, but not modification of the novel site. Together, the results suggest that the snoRNA hairpins function in a coordinated manner and that their interactions with pre-rRNA could be coupled.

Control of primary infection with hepatitis C virus (HCV) is associated with robust and broad T cell immunity. In contrast, chronic infection is characterized by weak T cell responses suggesting that an approach that boosts these responses could be a therapeutic advance. Saccharomyces cerevisiae is an effective inducer of innate and adaptive cellular immunity and we have generated recombinant yeast cells (GI-5005) that produce an HCV NS3-Core fusion protein. Pre-clinical studies in mice showed that GI-5005 induced potent antigen-specific proliferative and cytotoxic T cell responses that were associated with Th1-type cytokine secretion. In studies in which GI-5005 was administered up to 13 times, no detectable vector neutralization or induction of tolerance was observed. Prophylactic as well as therapeutic administration of GI-5005 in mice led to eradication of tumor cells expressing HCV NS3 protein. Immunotherapy with GI-5005 is being evaluated in chronic HCV infected individuals in a Phase 1 clinical trial.

Importance Purse-string suture is a closure method that purportedly reduces the scar area compared with second intention healing. Randomized clinical trials comparing these 2 methods appear to be limited or absent. Objective To determine if purse-string suture improves cosmetic outcome, healing time, and scar to defect area compared with second intention healing for circular defects on the trunk and extremities. Design, Setting, and Participants Prospective, 2-arm, randomized, evaluator-blinded clinical trial in a single-center outpatient academic dermatology center. Patients were eligible if they were older than 18 years, able to give informed consent, and had circular or oval postoperative defects larger than 8 mm on the trunk or extremities. Interventions For the purse-string treatment arm, wounds were sewn in circumferential fashion using polydiaxanone suture. Patients in the other treatment arm were allowed to heal by second intent. Main Outcomes and Measures The primary outcome measures were the mean total Patient and Observer Scar Assessment Scale (POSAS) scores ascertained from the patient and 2 blinded observers. Secondary outcomes included the ratio of scar to initial defect size, healing time, pain scores, and complication rates. Results Fifty-two patients were screened, and a total of 44 patients with 50 surgical sites were enrolled. Forty-two patients with 48 surgical sites completed the study. The mean total observer POSAS score was 18.38 for the purse-string group vs 19.91 for the secondary intention group, a nonsignificant difference (P = .41). Similarly, there were no significant differences for any of the following secondary outcome measures: mean total patient POSAS score (P = .96), mean scar-to-defect area (P = .61), and mean pain level at week 1 (P = .19). Statistical trends toward significance were seen in the mean healing time in favor of purse-string suture (P = .10) and scar relief, which favored second intention healing (P = .07). Conclusions and Relevance The purse-string suture results in similar cosmetic outcomes, scar sizes, and pain levels compared with second intention healing for circular or oval wounds on the trunk and extremities. A larger study might better define the potential differences in our secondary outcome measures of healing time and scar relief. Trial Registration clinicaltrials.gov Identifier:NCT02062866

Background Electrobrasion, like dermabrasion, is a method of surgical planing that is purported to improve postoperative scarring. Data regarding its benefits and harms relative to dermabrasion are absent. Objective To compare the efficacy and potential harms of electrobrasion and dermabrasion. Methods This was a pragmatic, randomized, double-blind, split-scar intervention in patients with suboptimal surgical outcomes. Half of the wound was randomized to treatment with dermabrasion and half to electrobrasion. At 3-month follow-up, both the patient and a blinded investigator evaluated the wound. Results Electrobrasion and dermabrasion reduced the mean scores of the Manchester Scar Scale 1·6 and 1·3 points from baseline, respectively (P = 0·0003). The difference between treatments was not significant (P = 0·08). Global cosmetic improvement by physician and patient assessment indicated clinical improvement for both procedures but did not demonstrate statistical significance between treatments (P = 0·57, P = 0·32 for physician and patient, respectively). Conclusions Both dermabrasion and electrobrasion improved scars, but there was no significant difference between the outcomes of the two procedures on several measures. Procedure time and bleeding time were significantly lower for electrobrasion. What's already known about this topic? Electrobrasion has been described as an alternative means of surgical planing, but has not been evaluated in comparison with more traditional techniques. What does this study add? This study present the results of a randomized, controlled, split-scar intervention comparing the techniques of electrobrasion and dermabrasion in the treatment of a variety of suboptimal surgical scars. Electrobrasion is a viable alternative to dermabrasion, as outcomes did not differ significantly post-treatment as evaluated using a standardized scar scale. Procedure time and bleeding time were favorable for electrobrasion. © 2014 British Association of Dermatologists.

Background: The set-back suture, an absorbable dermal suturing technique, purportedly improves wound eversion and cosmetic outcomes. Objective: We sought to conduct a split-wound, prospective, randomized study to compare the cosmetic outcome and wound eversion achieved with the set-back suture and the buried vertical mattress suture (BVMS). Methods: A total of 46 surgical elliptical wounds were randomized to subcuticular closure with the set-back suture on half and the BVMS on the other. Maximum eversion height and width were measured immediately postoperatively. At 3 months, 2 blinded observers evaluated each scar using a 7-point Likert physician global scar assessment scale. Subjects and observers also completed the validated Patient and Observer Scar Assessment Scale, where a score of 6 represents normal-appearing skin and 60 represents worst imaginable scar. Results: In all, 42 subjects completed the study. The set-back suture provided statistically significant wound eversion. On the Likert scale, observers rated the set-back suture side 1 point better than the BVMS side. Both patient and observer total Patient and Observer Scar Assessment Scale scores were significantly lower for the set-back suture side (subject mean 13.0 ± 8.7 vs 16.2 ± 12.0 [P = .039]; observer mean 24.5 ± 10.4 vs 27.7 ± 13.6 [P = .028], respectively). Limitations: Single institution experience and relatively short follow-up are limitations. Conclusion: The set-back suture provides superior wound eversion and better cosmetic outcomes than the BVMS. © 2014 American Academy of Dermatology, Inc.

In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA. A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

The treatment of facial cysts often entails some thorny decision making for the dermatologist. We offer a review of several approaches for their removal or modification and examine the outcome evidence for some common techniques and some that are seen less often. Finally, we offer our recommendations based on this trial evidence.

Background Critical to the accuracy of Mohs surgery is the ability to maintain proper orientation of excised tissue with respect to the surrounding skin. Several techniques have been described for maintaining this orientation, although no prior investigations directly compare these techniques. Objective To compare the incidence of tissue orientation loss resulting from inability to identify skin score marks with that occurring from failure to identify marks made using a gentian violet marker during Mohs micrographic surgery (MMS). We also sought to determine the incidence of scars resulting from skin scoring. Materials and Methods Patients undergoing MMS were prospectively randomized to have their tissue margins oriented using light scoring using a scalpel versus marking them using a gentian violet marker. Incidence of scoring scars and tissue orientation loss were the primary outcome measures. Results Data were analyzed for 101 tumors. There were no instances of tissue orientation loss in the scalpel or marker arms, nor were there any visible score mark scars at follow-up. Conclusion Incidence of excessive scars resulting from lightly scored tissue or loss of tissue orientation caused by lost gentian violet markings appears to be low. Both methods worked well within the confines of this study.

Immunotherapy for cancer represents an attractive therapeutic target because of its specificity and lack of toxicity, but products investigated so far have been limited by neutralisation, complexity of manufacturing and requirement for patient-specific products. Recombinant yeast cells are capable of stimulating the immune system to produce highly specific and potent cellular responses against target protein antigens with little toxicity. Data from animal models suggest that Tarmogens™ (yeast-based immunotherapeutics) can elicit protective immunity against xenografted and chemically induced tumours. This concept is now being tested in a Phase I trial in patients with colorectal, pancreatic and non-small cell lung cancers.

Drug target discovery and validation are complex processes that require significant resource investments and impose a substantial economic burden on the pharmaceutical industry. Technologies that accelerate or enhance the precision of target selection are, therefore, in high demand. Traditional antisense and RNA interference (RNAi) technologies are powerful tools with applications in multiple phases of drug target discovery and validation. These approaches elicit potent and highly selective cleavage of a target mRNA, permitting evaluation of the role of the corresponding protein based on a loss-of-function phenotype. Incorporation of these technologies into high-throughput screens, in vitro biological assays and in vivo disease models provides valuable insight into gene function. Efforts are also underway to develop these agents as drugs. This review presents recent studies involving antisense and RNAi, and discusses how these technologies are facilitating target selection at various stages of the drug development process.

One of the oldest questions in RNA science is the role of nucleotide modification. Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications. Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding. Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations. Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others. The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed.

Background: While mutations in the ras gene and corresponding product are known to be etiologic in a number of human cancers, efforts to block the mutated protein have been largely unsuccessful leading to its characterization as “undruggable.” We have taken an alternative approach involving generation of T cell responses to the mutated protein. GI-4000 is a series of proprietary immunotherapeutics designed to target cells with activating ras mutations using heat-killed Saccharomyces cerevisiae yeast (named Tarmogens: Targeted Molecular Immunogens) genetically engineered to express Ras G12 and Q61 mutations. Tarmogens activate antigen-specific T cell-mediated immune responses that kill target cells expressing a number of cancer antigens including mutated Ras. Activating mutations in ras occur in > 90% of pancreas cancer cases and >20% cases of NSCLC. GI-4000 has been evaluated in phase 2 trials in both these indications. GI-4000 has demonstrated: i) protection in a murine model of lung cancer (1), ii) in the pancreas trial, improvements in median RFS and OS vs placebo in subjects with a favorable proteomic signature (2), iii) also in the pancreas trial, 3 month improvement in median OS (p=NS) in R1 subjects, with 5 month improvement for immune responders (2), iv) improved OS in subjects with NSCLC treated with GI-4000 compared to case matched controls: HR=0.577, p=NS. In addition, Tarmogens specific for other oncologic targets decreased human regulatory T cells (Tregs) in vitro with a reciprocal increase in effector T cells (3). Here we discuss immunologic outcomes in R0 subjects enrolled in the pancreas cancer trial. Methods: In the pancreas cancer study 176 subjects with Ras mutant+ adenocarcinoma of the pancreas post resection were randomized 1:1 to GI-4000/gemcitabine or placebo/gemcitabine (stratified by resection status: R0/R1). Three weekly injections of GI-4000 or placebo were followed by 6 cycles of gemcitabine 1000 mg/m2 iv (day 1, 8, 15, then every 28 days). Monthly GI-4000 or placebo were administered on gemcitabine off-weeks and continued monthly until disease recurrence, intolerable toxicity or death. R0 subjects (n=102) with adequate blood samples available from timepoints throughout the study were assayed for immune response by interferon-γ (IFNγ) ELISpot assay using Ras peptide pools containing the G12 mutation present in the subject's tumor and a mismatched peptide set identical to the mutation-specific set except at G12. Frequencies of Tregs at baseline and pre-gemcitabine were measured by flow cytometry (n=76). Results: In contrast to the R1 group, there was no increase in Ras mutation-specific IFNγ responses in the R0 GI-4000 group compared to placebo: 16/52 (30.8%) vs 22/50 (44.0%) based on pre-specified criteria. However, naive Tregs (CD4+/CD45RA+/Foxp3low) in the GI-4000 treated group were significantly decreased compared to placebo: 11/42 (26.2%) vs 3/34 (8.8%) subjects had a >2-fold decrease in this fraction (p=0.048 Fisher's exact test). IFNγ responses and OS were strikingly influenced by G12 mutation and associations of specific Ras mutations with outcome will be discussed. Conclusions: GI-4000 decreases frequencies of Tregs, which could be an important attribute for an immunotherapeutic in the treatment of cancer. GI-4000 also generates mutation-specific immune responses and appears to have clinical activity in pancreas cancer and NSCLC. Immune targeting of the activating mutation may be a promising approach to Ras mutated cancers. References: 1. Lu Y., et al. Cancer Research 64: 5084-88, 2004. 2. Richards D.A., et al. ESMO, Vienna, Austria, Sept 2012. 3. Cereda V., et al. Vaccine 29: 4992-99, 2011. Citation Format: Claire Coeshott, Tom Holmes, Alicia Mattson, Tom King, Zhimin Guo, Allen Cohn, Timothy C. Rodell. Immune responses to mutated Ras - development of a yeast-based immunotherapeutic. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A28. doi: 10.1158/1557-3125.RASONC14-A28

TPS3127 Background: Saccharomyces cerevisiae has been genetically modified to express CEA protein and developed under a CRADA with GlobeImmune/NCI as a heat-killed immune-stimulating, therapeutic cancer vaccine (GI-6207). A phase I study with GI-6207 demonstrated safety, biomarker stabilization and enhanced immune response in some patients. CEA is over-expressed in multiple malignancies, including medullary thyroid cancer (MTC). Two therapies recently approved by the FDA for metastatic MTC (vandetanib, cabozantinib) come with toxicity and should be reserved for symptomatic/progressive disease. However, a large population of asymptomatic MTC patients has small tumor burden and/or disease that is more indolent. The standard management of these patients is observation. Preliminary data suggest that tumor growth measured by the rate of CEA and calcitonin increase can be quantified in a 3-6 months. Retrospective data from prostate cancer studies suggest vaccines can alter growth rates within 3-4 months. We hypothesize that GI-6207 can alter tumor growth rates in MTC and impact long-term outcome. Methods: A phase II study will evaluate the effect of GI-6207 onthe rates ofincrease in calcitonin in metastatic MTC. 34 patients with minimally symptomatic, radiographically evaluable, metastatic MTC will be randomized 1:1. Arm A will receive vaccine for a year from the time of enrollment. Arm B will receive vaccine after 6 months of surveillance. GI-6207 will be administered subcutaneously at 4 sites (10 yeast units/site), every 2 weeks for 3 months, then monthly up to 1 year. The primary endpoint will compare the effect of GI-6207 on calcitonin kinetics between the vaccine and surveillance arms in the first 6 months. Secondary endpoints include immunologic responses (including antigen-specific T cell responses), objective responses, time to progression, and changes in CEA kinetics. If this trial can prospectively demonstrate that vaccines can alter tumor growth rates, and if such changes are associated with clinical outcomes, then changes in tumor growth rates may become a clinical metric to evaluate vaccine efficacy in MTC and other populations.

Background: Tivozanib, a potent, selective, long-half-life tyrosine kinase inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, has demonstrated antitumor activity in a Phase II study in patients with renal cell carcinoma (RCC), and is currently being studied in clinical trials in patients with RCC and other solid tumors. The goal of this study was to investigate the effect of food on the pharmacokinetics (PK) of a single 1.5 mg dose of tivozanib in healthy subjects. Methods: This was a single-center, open-label, randomized, two-period, crossover Phase I trial. Subjects were admitted to the clinical research unit (CRU) 1 day before dosing, fasted ∼10 hours, and were randomized to fed (standard high-fat breakfast)/fasted or fasted/fed sequence. In each phase of the sequence, subjects received a single oral dose of tivozanib 1.5 mg with a 6-week washout period between doses. Subjects remained at the CRU for at least 48 hours post dose for blood sample collection and safety monitoring, and were assessed on an outpatient basis for up to 504 hours post dose. PK data were analyzed by non-compartmental methods. The effect of food on PK was assessed using standard criteria for bioequivalence based on the exposure parameters AUC0-α and Cmax. If the 90% confidence intervals for the fed/fasted AUC0-α and Cmax fell within the range of 80% to 125%, it was concluded that food had no effect on exposure. Results: Thirty healthy volunteers were enrolled (19M/11F; mean age 39 years [range 22-53 years]). There was no significant difference in AUC0-α of serum tivozanib between the fed and fasted states (107.4%; Table 1). Food caused a significant decrease in serum tivozanib levels vs fasted state (Cmax: 77.5%). Conclusions: These results indicate that dosing tivozanib with food decreases maximal concentrations by ∼ 23%, but does not affect overall exposure. As tivozanib is dosed chronically in oncology patients and accumulates ∼6 to 7 times single-dose levels when at steady-state, these results are not likely to affect dosing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 752. doi:1538-7445.AM2012-752