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Purpose: The goal of this study was to investigate the therapeutic potential of a novel immunotherapy strategy resulting in immunity to localized or metastatic human papillomavirus 16–transformed murine tumors. Experimental Design: Animals bearing E7-expressing tumors were coimmunized by lymph node injection with E7 49-57 antigen and TLR3-ligand (synthetic dsRNA). Immune responses were measured by flow cytometry and antitumor efficacy was evaluated by tumor size and survival. In situ cytotoxicity assays and identification of tumor-infiltrating lymphocytes and T regulatory cells were used to assess the mechanisms of treatment resistance in bulky disease. Chemotherapy with cyclophosphamide was explored to augment immunotherapy in late-stage disease. Results: In therapeutic and prophylactic settings, immunization resulted in a considerable expansion of E7 49-57 antigen–specific T lymphocytes in the range of 1/10 CD8+ T cells. The resulting immunity was effective in suppressing disease progression and mortality in a pulmonary metastatic disease model. Therapeutic immunization resulted in control of isolated tumors up to a certain volume, and correlated with antitumor immune responses measured in blood. In situ analysis showed that within bulky tumors, T-cell function was affected by negative regulatory mechanisms linked to an increase in T regulatory cells and could be overcome by cyclophosphamide treatment in conjunction with immunization. Conclusions: This study highlights a novel cancer immunotherapy platform with potential for translatability to the clinic and suggests its potential usefulness for controlling metastatic disease, solid tumors of limited size, or larger tumors when combined with cytotoxic agents that reduce the number of tumor-infiltrating T regulatory cells. (Clin Cancer Res 2009;15(19):6167–76)

Background: Gym equipment surfaces are known to harbor a range of contaminants due to the wide range of community use of the equipment. Certain gym equipment undergoes daily sanitation, however many other equipment surfaces do not. This study measures the levels of contamination on certain gym equipment surfaces at an educational institute gym facility and determines the contamination levels after disinfectant wipes are applied. Methods: The method to obtain the data was determined by the use of the Hygiena Systemsure II ATP analyzer in conjunction with Hygiena Ultrasnap ATP surface swabs. Gym equipment (barbells, dumbbells, machine handles, cable attachments) and other surfaces (benches, floor mats) were swabbed subsequently after a random gym patron had used the equipment to capture an accurate representation of the cleanliness of the surfaces. Disinfectant wipes were then applied to the same area before being swabbed again to determine contamination levels after disinfection. Results: The results demonstrated a statistically significant difference in the reduction of ATP levels with the use of disinfectant wipes with a p-value of 0.00001 at α=0.05. Alpha error was highly unlikely with a p-value being that low. Power was 99.9%, therefore there is a strong likelihood that we are correctly rejecting the null hypothesis. Conclusion: The study can conclude that disinfectant wipes do make a significant difference in surface cleanliness levels. Equipment that does not undergo routine cleaning such as the equipment used by the hands carry a much higher contamination rate than the body contact surfaces. Gym patrons should disinfect all body contact surfaces prior to use to reduce the risk of getting an infectious disease.

Physicians and nurses were included in the study in order to see whether role ambiguity for medical social workers is related to the differences in role expectations. It was found that medical social workers had different expectations of their roles from those of physicians and nurses. A clear difference was also identified between the lines of authority in terms of medical social workers’ role relations with physicians and nurses. Role ambiguity of medical social workers did not merely arise out of misunderstanding or misperception, it was also affected by authority structure.

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

Background Ventilator associated pneumonia (VAP) in the intensive care unit (ICU) has been shown to be associated with significant morbidity and mortality. 1–3 It has been reported to affect between 9 and 27% of intubated patients receiving mechanical ventilation. 4–6 Objective A meta-analysis was undertaken to combine information from published studies of the effect of subglottic drainage of secretions on the incidence of ventilated associated pneumonia in adult ICU patients. Data sources Studies were identified by searching MEDLINE (1966 to January 2011), EMBASE (1980–2011), and CINAHL (1982 to January 2011). Review methods Randomized trials of subglottic drainage of secretions compared to usual care in adult mechanically ventilated ICU patients were included in the meta-analysis. Results Subglottic drainage of secretions was estimated to reduced the risk of VAP by 48% (fixed-effect relative risk (RR)=0.52, 95% confidence interval (CI), 0.42–0.65). When comparing subglottic drainage and control groups, the summary relative risk for ICU mortality was 1.05 (95% CI, 0.86–1.28) and for hospital mortality was 0.96 (95% CI, 0.81–1.12). Overall subglottic drainage effect on days of mechanical ventilation was −1.04 days (95% CI, −2.79–0.71). Conclusion This meta-analysis of published randomized control trials shows that almost one-half of cases of VAP may be prevented with the use of specialized endotracheal tubes designed to drain subglottic secretions. Time on mechanical ventilation may be reduced and time to development of VAP may be increased, but no reduction in ICU or hospital mortality has been observed in published trials.

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

Unplanned admission to the intensive care unit has been shown to significantly increase the risk of inhospital mortality. Medical advances and increased expectations have resulted in a greater number of very elderly patients (80 years and over) being admitted to the ICU. The risk of in-hospital death associated with unplanned admission to the ICU in very elderly patients has not been clearly defined.To estimate the risk of in-hospital mortality associated with unplanned admission to the ICU in patients aged 80 years and over.Retrospective review of an adult intensive care database. The setting was Liverpool Hospital, a large teaching hospital in Sydney, Australia, with a 28-bed ICU that has about 2000 admissions per year. We analysed data on very elderly patients (n = 1680), aged 80 years or more, admitted to the ICU between 1 January 1997 and 31 December 2007.Baseline risk factors for inhospital mortality.Mortality among patients with unplanned ICU admissions was 47%, compared with 25% in patients with planned admissions (adjusted rate ratio [RR], 1.92 [95% CI, 1.59-2.32]). An estimated 50% of the overall risk of inhospital death among very elderly patients was attributable to a combination of unplanned admission to the ICU, the presence of at least one comorbid condition, acute renal failure and respiratory failure requiring intubation.Unplanned admission to the ICU increases the risk of in-hospital mortality in very elderly patients. At least 50% of the risk of in-hospital death in this age group is attributable to a combination of unplanned ICU admission, comorbidity (≥1 comorbid condition), acute renal failure and respiratory failure.

Readmission to intensive care during the same hospital stay has been associated with a greater risk of in-hospital mortality and has been suggested as a marker of quality of care. There is lack of published research attempting to develop clinical prediction tools that individualise the risk of readmission to the intensive care unit during the same hospital stay.To develop a prediction model using an inception cohort of patients surviving an initial ICU stay.The study was conducted at Liverpool Hospital, Sydney. An inception cohort of 14 952 patients aged 15 years or more surviving an initial ICU stay and transferred to general wards in the study hospital between 1 January 1997 and 31 December 2007 was used to develop the model. Binary logistic regression was used to develop the prediction model and a nomogram was derived to individualise the risk of readmission to the ICU during the same hospital stay.Readmission to the ICU during the same hospital stay.Among members of the study cohort there were 987 readmissions to ICU during the study period. Compared with patients not readmitted to the ICU, patients who were readmitted were more likely to have had ICU stays of at least 7 days (odds ratio [OR], 2.2 [95% CI, 1.85- 2.56]); non-elective initial admission to the ICU (OR, 1.7 [95% CI, 1.44-2.08]); and acute renal failure (OR, 1.6 [95% CI, 0.97-2.47]). Patients admitted to the ICU from the operating theatre or recovery ward had a lower risk of readmission to ICU than those admitted from general wards, the emergency department or other hospitals. The maximum error between observed frequencies and predicted probabilities of readmission to ICU was estimated to be 3%. The area under the receiver operating characteristic curve of the final model was 0.66.We have developed a practical clinical tool to individualise the risk of readmission to the ICU during the same hospital stay in patients who survive an initial episode of intensive care.

The use of adhesives to retain facial prostheses remains controversial. This controversy arises in part from a lack of information on the biomechanical performance of this group of materials. No standard exists for maxillofacial prosthetic adhesives. This study describes the rationale for and design of an apparatus for testing maxillofacial and other skin adhesives. The apparatus provides for tensile, torsion, and combined tensile-torsion tests in a hard machine test. The results of the calibration studies of the apparatus are discussed.

Elevated Programmed Death-1 (PD-1) expression can inhibit T cell activity and is a potential barrier to achieving persisting and optimal immunity via therapeutic vaccination. Using a direct lymph node-targeted vaccination procedure that enabled uncoupling of synthetic peptide (signal 1, TCR-mediated) and adjuvant (signal 2, non-TCR-mediated), we evaluated the impact of varied doses of Toll-like receptor (TLR)-9 ligand CpG oligodeoxynucleotide (ODN) adjuvant on epitope-specific CD8(+) T cell-associated PD-1 expression. Peptide vaccination without adjuvant yielded CD8(+) T cells with significantly elevated PD-1 expression. This conferred impaired function ex vivo, but was reversible by antibody-mediated PD-1 blockade. By comparison, peptide vaccination with escalating doses of CpG ODN adjuvant yielded higher magnitudes of CD8(+) T cells with progressively lower PD-1 expression and greater ex vivo function. CpG ODN adjuvant in context of titrated peptide doses for vaccination yielded the lowest overall PD-1 expression levels, demonstrating that fine-tuning both TCR-independent (adjuvant dose) and -dependent (antigen dose) stimuli can synergize to co-regulate PD-1 expression on epitope-specific CD8(+) T cells. These data hint at strategies to elicit PD-1(low) CD8(+) T cells using TLR-9 ligand adjuvants, and also shed light on the PD-1-regulated homeostasis of CD8(+) T cells.

Summary Aim Although unplanned admissions to the intensive care unit (ICU) are associated with poorer prognoses, there is no published prognostic tool available for predicting this risk in an individual patient. We developed a nomogram for calculating the individualised absolute risk of unplanned ICU admission during a hospital stay. Method Hospital administrative data from a large district hospital of consecutive admissions from 1 January 2000 to 31 December 2006 of aged over 14 years was used. Patient data was extracted from 94,482 hospital admissions consisted of demographic and clinical variables, including diagnostic categories, types of admission and time and day of admission. Multivariate logistic regression coefficients were used to develop a predictive nomogram of individual risk to patients admitted to the study hospital of unplanned ICU admission. Results A total of 672 incident unplanned ICU admissions were identified over this period. Independent predictors of unplanned ICU admissions included being male, older age, emergency department (ED) admissions, after-hour admissions, weekend admissions and six principal diagnosis groups: fractured femur, acute pancreatitis, liver disease, chronic airway disease, pneumonia and heart failure. The area under the receiver operating characteristic curve was 0.81. Conclusion The use of a nomogram to accurately identify at-risk patients using information that is readily available to clinicians has the potential to be a useful tool in reducing unplanned ICU admissions, which in turn may contribute to the reduction of adverse events of patients in the general wards.

Despite the recent advances made in the treatment and management of B cell malignancies, these diseases are not curable and overall survival is limited. Bruton's Tyrosine Kinase (BTK) is a member of the Tec family of intracellular kinases first identified for its signaling via the B-cell Receptor (BCR) and its role in the immune system. More recently, BTK was found to play an important role in B cell malignancies and select solid tumors. Preclinical and clinical results with selective irreversible BTK inhibitors provide validation for BTK as a therapeutic target in B cell malignancies. Aiming to leverage the contribution of the BTK signaling pathway to tumor growth, and its role in progression and drug resistance, we have developed a series of relatively selective, reversible, small molecule BTK inhibitors and evaluated their activity in enzyme, cell-based, and in vivo studies. Data obtained with the orally available MKC4659 compound illustrates our findings. In biochemical assays, compound MKC4659 demonstrated a relatively select targeting profile focused on a narrow group of PTKs including significant activity against BTK with IC50 less than 25 nM. In cellular assays the compound demonstrated significant in vitro potency against B cell lymphoma cell lines, inhibiting the growth of several B cell tumor cell lines including ones unresponsive to currently known BTK inhibitors. Importantly, MKC4659 showed a differential effect on B cell lymphoma, with no significant activity detected in control cells lacking detectable BTK expression. In vitro mode of action studies demonstrated that MKC4659 induces apoptosis and PARP cleavage in B cell lymphoma but not in control cells. Assays evaluating the in vitro on-target effect of compounds showed significant inhibition of the B cell receptor-mediated activation of the BTK pathway. In addition to inhibiting the phosphorylation of BTK, MKC4659 inhibited the phosphorylation of PLCγ in several B cell lymphoma cell lines. With in vitro potency demonstrated, and PK and ADMET profiles amenable to in vivo dosing, MKC4659 was evaluated for in vivo efficacy in a xenograft model of B cell lymphoma. In vivo dosing of MKC4659 inhibited growth of DOHH2 xenograft tumors in a dose dependent manner. In summary, our team has identified BTK pathway inhibitors with demonstrated on-target and anti-tumor activity in cellular assays, and efficacy in a preclinical model of B cell malignancy. This effort provides a platform for compound development and evaluation for the treatment of hematologic malignancies. Optimization efforts on the MKC4659 series are ongoing and have yielded potent and drug-like preclinical candidates that are now moving into advanced animal studies. Citation Format: Mary Faris, Uriel M. Malyankar, Victor Tam, Colleen Schweitzer, Diljeet Joea, Alexis Mollard, Bret Stephens, Steven L. Warner, David J. Bearss, Qingping Zeng. Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2013-LB-303

Abstract 133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

Abstract 4074 Aberrant protein folding results in the accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER), which in turn triggers ER stress followed by unfolded protein response (UPR), an adaptive response against ER stress. Since multiple myeloma (MM) cells have high protein synthesis, they are sensitive to ER stress and require strict ER quality control for cell survival. Upon UPR, IRE1α is activated by auto-phosphorylation resulting in activation of its endoribonuclease domain to splice XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to XBP1 spliced form (XBP1s: active). Since XBP1 is a transcription factor regulating genes which are responsible for protein folding and ER associated degradation (ERAD), IRE1α-XBP1 pathway acts as a pro-survival signaling pathway under the UPR condition. In this study, we examined whether IRE1α-XBP1 pathway is a potential novel therapeutic option in MM. We first examined IRE1α expression and confirmed its expression in all MM cell lines. In contrast, XBP1s was not detected by RT-PCR in most cell lines except in for RPMI8226 cells. To assess biologic significance of IRE1α in MM cell, we knock-downed its expression using shRNA and found that downregulation of IRE1α inhibited MM cell growth, indicating that IRE1α has a crucial role in MM cell survival. We next examined the impact of inhibition of XBP1 splicing by a small molecule IRE1α endoribonuclease inhibitor MKC-3946 (Mannkind, Valencia CA) in MM cells in vitro. As expected, MKC-3946 significantly inhibited tunicamycin-induced XBP1s without affecting phosphorylation of IRE1α. MKC-3946 induced only modest cytotoxicity in MM cell lines without toxicity in normal mononuclear cells from healthy donors; however, it significantly enhanced cytotoxicity in combination with bortezomib or 17-AAG. Both bortezomib and 17-AAG induced ER stress evidenced by induction of XBP1s; conversely, MKC-3946 blocked XBP1s triggered by these agents. Furthermore, apoptosis induced by these agents was enhanced with MKC-3946 associated with increased CHOP, which is a known pro-apoptotic protein induced in uncompensated ER stress condition. Importantly, MKC-3946 enhanced the cytotoxicity of bortezomib or 17-AAG in INA6 cells, even in the presence of increased IL-6 or bone marrow stromal cells. Finally, MKC-3946 was active inhibiting XBP1 splicing in a model of ER stress and significantly inhibited growth of RPMI8226 plasmacytoma in a xenograft murine model when used in combination with a low dose of bortezomib. Taken together, our results demonstrate that inhibition of XBP1 splicing by blockade of IRE1α is a promising therapeutic option in MM. Disclosures: Blumenthal: Mannkind Corporation: Employment, Equity Ownership. Tam:Mannkind Corporation: Employment, Equity Ownership. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Zeng:Mannkind Corporation: Employment, Equity Ownership. Patterson:Mannkind Corporation: Employment, Equity Ownership. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

The purpose of this study was to advance a novel immunotherapy platform technology that would achieve objective clinical response and allow subsequent elucidation of immune correlates of clinical responses. To this aim, we tested an immunotherapy approach encompassing sequential intra-lymph node administration of DNA vectors and peptide analogues to elicit multivalent CD8+ T cell immunity against cancer cell and stromal antigens. This included the assessment of safety, immune response and clinical outcome following treatment with two investigational regimens in patients with metastatic solid tumors such as melanoma, prostate, and renal clear cell carcinoma. Strikingly, T cell transcriptomal analysis by gene array and flow cytometry analysis in a preclinical model showed that DNA vaccination induced a distinct immunophenotype, compared to peptide vaccination. DNA vaccine-primed CD8+ T cells showed a coordinated, lower expression of co-inhibitory molecule PD-1 and retention of the lymph node homing molecule CD62L, leading to a significant persistence of antigen-specific T cells, cellular expansion and functional conversion to effector cells, upon peptide boosting. Secondly, both objective tumor regression and elevation of antigen-specific T cells were observed in immunized patients, although there was no strict correlation between these two outcomes. Instead, there was a disease stage dependency of the presence of T cells specific for Melan A / MART-1 in melanoma patients, with all stage IV M1a (lymphatic metastatic disease) patients exhibiting pre-existing T cells and responding to treatment through durable tumor regression. Significantly, T cells specific for both immunizing antigens, Melan A/MART-1 and Tyrosinase, were identifiable within regressing tumor lesions in these M1a patients. In contrast, the preponderance of pre-existing Melan A/MART-1 T cells in melanoma patients with visceral metastatic disease was less than 50% and no apparent tumor regression upon administration was observed. In a second parallel trial, co-immunization against PRAME and PSMA generated clinical response through disease stabilization in several patients with carcinoma and was mirrored by a persisting T cell immunity. In summary, these results lead to three major conclusions: first, design of synthetic active immunotherapies affording objective tumor regression is possible, thereby refuting the paradigm that such approaches can be effective only in earlier disease stage; second, a systemic, hypothesis-generating approach must be employed to uncover appropriate biomarker correlates; and third, DNA-based vaccination has the potential of being translated to safe and effective therapies for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2398.