Uncoupling CD4+ TIL-Mediated Tumor Killing from JAK-Signaling in Melanoma.

PURPOSE: Impaired MHCI-presentation and insensitivity to immune effector molecules are common features of immune checkpoint blockade (ICB)-resistant tumors and can be, respectively, associated with loss of ß2 microglobulin (B2M) or impaired IFNγ signaling. Patients with ICB-resistant tumors can respond to alternative immunotherapies, such as infusion of autologous tumor-infiltrating lymphocytes (TIL). CD4+ T cells can exert cytotoxic functions against tumor cells; however, it is unclear whether CD4+ T-cell responses can be exploited to improve the clinical outcomes of patients affected by ICB-resistant tumors. EXPERIMENTAL DESIGN: Here, we exploited CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing to reproduce immune-resistant tumor phenotypes via gene knockout (KO). To determine the role of cytotoxic CD4+ TILs in ICB-resistant tumors, we investigated CD4+ TIL-mediated cytotoxicity in matched pairs of TILs and autologous melanoma cell lines, used as a model of patient-specific immune-tumor interaction. Around 40% of melanomas constitutively express MHC Class II molecules; hence, melanomas with or without natural constitutive MHC Class II expression (MHCIIconst+ or MHCIIconst-) were used. RESULTS: CD4+ TIL-mediated cytotoxicity was not affected by B2M loss but was dependent on the expression of CIITA. MHCIIconst+ melanomas were killed by tumor-specific CD4+ TILs even in the absence of IFNγ-mediated MHCII upregulation, whereas IFNγ was necessary for CD4+ TIL-mediated cytotoxicity against MHCIIconst- melanomas. Notably, although tumor-specific CD4+ TILs did not kill JAK1KO MHCIIconst- melanomas even after IFNγ stimulation, sensitivity to CD4+ TIL-mediated cytotoxicity was maintained by JAK1KO MHCIIconst+ melanomas. CONCLUSIONS: In conclusion, our data indicate that exploiting tumor-specific cytotoxic CD4+ TILs could help overcome resistance to ICB mediated by IFNγ-signaling loss in MHCIIconst+ melanomas. See related commentary by Betof Warner and Luke, p. 3829.

Case Report: First longitudinal study of a patient with CALR positive clonal hematopoiesis of indeterminate potential developing into pre-fibrotic myelofibrosis.

Initial diagnosis of overt myeloproliferative neoplasms (MPNs) represents the juncture during clonal evolution when symptoms or complications prompt an afflicted individual to seek medical attention. In 30-40% of the MPN subgroups essential thrombocythemia (ET) and myelofibrosis (MF), somatic mutations in the calreticulin gene (CALR) are drivers of the disease resulting in constitutive activation of the thrombopoietin receptor (MPL). In the current study, we describe a healthy CALR mutated individual during a 12 year follow-up from initial identification of CALR clonal hematopoiesis of indeterminate potential (CHIP) to the diagnosis of pre-MF. The pre-diagnostic exponential development dynamics of the malignant clone demonstrated close correlation with the platelet counts, neutrophil-to-lymphocyte (NLR) ratio, and inversely correlated to hemoglobin and erythrocyte counts. Backward extrapolation of the growth rate indicated the potential for discovery of the malignant clone many years prior to presentation of overt disease, opening a window of opportunity for early treatment intervention. We did not find any additional mutations associated with MPNs and the current case report provides novel information regarding the development of a driver mutation and the association with blood cell counts prior to clinical manifestation of symptoms suggesting that pre-diagnostic dynamics may supplement future diagnostic criteria for early diagnosis and intervention in MPN patients.

Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

ABO blood types and sepsis mortality.

BACKGROUND: We aimed to determine if the ABO blood types carry different risks of 30-day mortality, acute kidney injury (AKI), and endothelial damage in critically ill patients with sepsis. This was a retrospective cohort study of three independent cohorts of critically ill patients from the United States and Scandinavia consisting of adults with septic shock. We compared the 30-day mortality across the blood types within each cohort and pooled the results in a meta-analysis. We also estimated the incidence of AKI and degree of endothelial damage, as measured by blood concentrations of soluble thrombomodulin and syndecan-1. RESULTS: We included 12,342 patients with severe sepsis. In a pooled analysis blood type B carried a slightly lower risk of 30-day all-cause mortality compared to non-blood type B (adjusted HR 0.88; 95%-CI 0.79-0.98; p = 0.02). There was no difference in the risk of AKI. Soluble thrombomodulin and syndecan-1 concentrations were lower in patients with blood type B and O compared to blood type A, suggesting less endothelial damage. CONCLUSION: Septic patients with blood type B had less endothelial damage, and a small reduction in mortality. The exposure is, however, unmodifiable.

High frequencies of circulating memory T cells specific for calreticulin exon 9 mutations in healthy individuals.

Mutations in exon 9 of the calreticulin gene (CALR) frequently occur in patients with chronic myeloproliferative neoplasms (MPN). Patients exhibit spontaneous cellular immune responses to epitopes derived from the mutant CALR C-terminus, and CALR-mutant-specific T cells recognize autologous CALR-mutant malignant cells. This study investigated whether CALR-mutant-specific T cells occur naturally in CALRwt MPN-patients and in healthy individuals. Specific immune responses against epitopes in the mutant CALR peptide sequence were detected in both CALRwt MPN-patients and in healthy individuals. Healthy donors displayed more frequent and stronger CALR-mutant specific T-cell responses compared to the responses identified in CALR-mutant MPN-patients. Several T-cell responses were identified in healthy donors directly ex vivo. Importantly, by running functional analyses on live-sorted immune cells from healthy donors, we showed that circulating CALR-mutant-specific immune cells are T-memory cells. These findings suggest, that healthy individuals acquire a CALR exon 9 mutation, but the immune system reacts and clears the mutant cells, and during this reaction generates CALR-mutant specific T-memory cells. We believe that these findings provide the evidence for tumor immune surveillance in MPN.

Predicting recovery from acute kidney injury in critically ill patients: development and validation of a prediction model.

OBJECTIVE: Intensive care unit (ICU) patients with acute kidney injury (AKI) who recover kidney function within 28 days experience less severe chronic kidney impairment and have increased long term survival. The aims of this study were to develop and validate a risk prediction model to identify these patients. DESIGN: Observational study with development and validation of a risk prediction model. SETTING: Nine academic ICUs in Denmark. PARTICIPANTS: Development cohort of critically ill patients with AKI at ICU admission from the Procalcitonin and Survival Study cohort (n = 568), validation cohort of adult patients with AKI admitted to two university hospitals in Denmark in 2012-13 (n = 766). INTERVENTIONS: None. MAIN OUTCOME MEASURES: Recovery of kidney function was defined as living for 5 consecutive days with no renal replacement therapy and with creatinine plasma levels below 1.5-fold the levels determined before ICU admission. RESULTS: A total of 266 patients (46.8%) recovered prior kidney function in the development cohort, and 453 patients (59.1%) in the validation cohort. The prediction model included elevation in creatinine, urinary output, sex and age. In the validation cohort, 69 patients (9.0%) had a predicted chance of recovery < 25%, and their observed rate of recovery was 21.5%. This observed rate of recovery was 81.7% among the 325 patients who had a predicted chance > 75%. The area under the receiver operations curves for predicting recovery in the validation cohort was 73.1%. CONCLUSION: We constructed and validated a simple model that can predict the chance of recovery from AKI in critically ill patients.

Low antigen dose formulated in CAF09 adjuvant Favours a cytotoxic T-cell response following intraperitoneal immunization in Göttingen minipigs.

The relationship between the antigen dose and the quality of an immune response generated upon immunization is poorly understood. However, findings show that the immune system is indeed influenced by the antigen dose; hence underlining the importance of correctly determining which dose to use in order to generate a certain type of immune response. To investigate this area further, we used Göttingen minipigs asan animal model especially due to the similar body size and high degree of immunome similarity between humans and pigs. In this study, we show that both a humoral and a cell-mediated immune (CMI) response can be generated following intraperitoneal immunization with tetanus toxoid (TT) formulated in the CAF09 liposomal adjuvant. Importantly, a low antigen dose induced more TT-specific polyfunctional T cells, whereas antigen-specific IgG production was observed upon high-dose immunization. Independent of antigen dose, intraperitoneal administration of antigen increased the amount of TT-specific cytotoxic CD8ß+ T cells within the cytokine-producing T-cell pool when compared to the non-cytokine producing T-cell compartment. Taken together, these results demonstrate that a full protein formulated in the CAF09 adjuvant and administered to pigs via the intraperitoneal route effectively generates a cytotoxic T-cell response. Moreover, we confirm the inverse relationship between the antigen dose and the induction of polyfunctional T cells in a large animal model. These finding can have implications for the design of upcoming vaccine trials aiming at establishing a cytotoxic T-cell response.

Prediction of non-recovery from ventilator-demanding acute respiratory failure, ARDS and death using lung damage biomarkers: data from a 1200-patient critical care randomized trial.

BACKGROUND: It is unclear whether biomarkers of alveolar damage (surfactant protein D, SPD) or conductive airway damage (club cell secretory protein 16, CC16) measured early after intensive care admittance are associated with one-month clinical respiratory prognosis. If patients who do not recover respiratory function within one month can be identified early, future experimental lung interventions can be aimed toward this high-risk group. We aimed to determine, in a heterogenous critically ill population, whether baseline profound alveolar damage or conductive airway damage has clinical respiratory impact one month after intensive care admittance. METHODS: Biobank study of biomarkers of alveolar and conductive airway damage in intensive care patients was conducted. This was a sub-study of 758 intubated patients from a 1200-patient randomized trial. We split the cohort into a "learning cohort" and "validating cohort" based on geographical criteria: northern sites (learning) and southern sites (validating). RESULTS: Baseline SPD above the 85th percentile in the "learning cohort" predicted low chance of successful weaning from ventilator within 28 days (adjusted hazard ratio 0.6 [95% CI 0.4-0.9], p = 0.005); this was confirmed in the validating cohort. CC16 did not predict the endpoint. The absolute risk of not being successfully weaned within the first month was 48/106 (45.3%) vs. 175/652 (26.8%), p < 0.0001 (high SPD vs. low SPD). The chance of being "alive and without ventilator ≥20 days within the first month" was lower among patients with high SPD (adjusted OR 0.2 [95% CI 0.2-0.4], p < 0.0001), confirmed in the validating cohort, and the risk of ARDS was higher among patients with high SPD (adjusted OR 3.4 [95% CI 1.0-11.4], p = 0.04)-also confirmed in the validating cohort. CONCLUSION: Early profound alveolar damage in intubated patients can be identified by SPD blood measurement at intensive care admission, and high SPD level is a strong independent predictor that the patient suffers from ARDS and will not recover independent respiratory function within one month. This knowledge can be used to improve diagnostic and prognostic models and to identify the patients who most likely will benefit from experimental interventions aiming to preserve alveolar tissue and therefore respiratory function. Trial registration This is a sub-study to the Procalcitonin And Survival Study (PASS), Clinicaltrials.gov ID: NCT00271752, first registered January 1, 2006.

Establishing the pig as a large animal model for vaccine development against human cancer.

Immunotherapy has increased overall survival of metastatic cancer patients, and cancer antigens are promising vaccine targets. To fulfill the promise, appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice, and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart, we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC, both important in human cancer development and progression, were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09, CASAC, Montanide ISA 51 VG, or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01, -1(*)07:02, -2(*)04:01, -2(*)05:02, and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer.

Reorienting the immune system in the treatment of cancer by using anti-PD-1 and anti-PD-L1 antibodies.

Physiologically, the programmed death 1 (PD-1) pathway is involved in limiting the killing of bystander cells during an infection and controlling autoimmunity. However, cancers exploit this system to avoid immune killing, and PD-1 ligand 1 and 2 (PD-L1 and PD-L2) expression on tumor cells, as well as PD-1 expression on tumor-infiltrating lymphocytes, have shown to be negative prognostic factors. Promising clinical results have been obtained by PD-1 pathway blockade in a range of cancers while still maintaining a manageable toxicity profile, and two anti-PD-1 antibodies are now approved by the US Food and Drug Administration (FDA) for the treatment of metastatic melanoma. As already shown with nivolumab and ipilimumab, the combination of PD-1 pathway blockade with other anticancer agents holds promise in the form of additive synergistic anticancer effects.

Oncogenic cancer/testis antigens: prime candidates for immunotherapy.

Recent developments have set the stage for immunotherapy as a supplement to conventional cancer treatment. Consequently, a significant effort is required to further improve efficacy and specificity, particularly the identification of optimal therapeutic targets for clinical testing. Cancer/testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor immunology and immune escape suggests that targeting oncogenic antigens may be beneficial, meaning that identification of cancer/testis antigens with oncogenic properties is of high priority. Recent work from our lab and others provide evidence that many cancer/testis antigens, in fact, have oncogenic functions, including support of growth, survival and metastasis. This novel insight into the function of cancer/testis antigens has the potential to deliver more effective cancer vaccines. Moreover, immune targeting of oncogenic cancer/testis antigens in combination with conventional cytotoxic therapies or novel immunotherapies such as checkpoint blockade or adoptive transfer, represents a highly synergistic approach with the potential to improve patient survival.

Profound endothelial damage predicts impending organ failure and death in sepsis.

Endothelial damage contributes to organ failure and mortality in sepsis, but the extent of the contribution remains poorly quantified. Here, we examine the association between biomarkers of superficial and profound endothelial damage (syndecan-1 and soluble thrombomodulin [sTM], respectively), organ failure, and death in sepsis. The data from a clinical trial, including critically ill patients predominantly suffering sepsis (Clinicaltrials.gov: NCT00271752) were studied. Syndecan-1 and sTM levels at the time of study enrollment were determined. The predictive ability of biomarker levels on death and organ failures during follow-up were assessed in Cox models adjusted for potential confounders including key organ dysfunction measures assessed at enrollment. Of the 1,103 included patients, 418 died. sTM levels at the time of enrollment independently predicted risk of death in adjusted models (hazard ratio [HR] [highest quartile > 14 ng/mL vs. lowest quartile < 7 ng/mL] 2.2 [95% confidence interval [CI]: 1.2-4.0], p = 0.02, respectively). Conversely, syndecan-1 levels failed to predict death (adjusted HR [> 240 vs. < 70 ng/mL] 1.0 [95% CI: 0.6-1.5], p = 0.67). sTM but not syndecan-1 levels at enrollment predicted risk of multiple organ failure during follow-up (HR [> 14 ng/mL vs. < 7 ng/mL] 3.5 [95% CI: 1.5-8.3], p = 0.005 and 2.0 [95% CI: 0.8-5.0], p = 0.1321, respectively). Profound damage to the endothelium independently predicts risk of multiple organ failure and death in septic patients. Our findings also suggest that the detrimental effect of profound endothelial damage on risk of death operates via mechanisms other than causing organ failures per se. Therefore, damage to the endothelium appears centrally involved in the pathogenesis of death in sepsis and could be a target for intervention.

Invasive Candida infections and the harm from antibacterial drugs in critically ill patients: data from a randomized, controlled trial to determine the role of ciprofloxacin, piperacillin-tazobactam, meropenem, and cefuroxime.

OBJECTIVE: Use of antibiotics in critically ill patients may increase the risk of invasive Candida infection. The objective of this study was to determine whether increased exposure to antibiotics is associated with increased prevalence of invasive Candida infection. DESIGN: Substudy using data from a randomized controlled trial, the Procalcitonin And Survival Study 2006-2010. SETTING: Nine multidisciplinary ICUs across Denmark. PATIENTS: A total of 1,200 critically ill patients. INTERVENTION: Patients were randomly allocated to either a "high exposure" antibiotic therapy (intervention arm, n = 604) or a "standard exposure" guided by current guidelines (n = 596). MEASUREMENTS AND MAIN RESULTS: Seventy-four patients met the endpoint, "invasive Candida infection," 40 in the high exposure arm and 34 in standard exposure arm (relative risk = 1.2; 95% CI, 0.7-1.8; p = 0.52). Among medical patients in the high exposure arm, the use of ciprofloxacin and piperacillin/tazobactam was 51% and 75% higher than in the standard exposure arm; no difference in antibiotic exposure was observed between the randomized arms in surgical patients. Among medical intensive care patients, invasive Candida infection was more frequent in the high exposure arm (6.2%; 27/437) than in standard exposure arm (3.3%; 14/424) (hazard ratio = 1.9; 95% CI, 1.0-3.6; p = 0.05). Ciprofloxacin used at study entry independently predicted invasive Candida infection (adjusted hazard ratio = 2.1 [1.1-4.1]); the risk gradually increased with duration of ciprofloxacin therapy: six of 384 in patients not exposed (1.6%), eight of 212 (3.8%) when used for 1-2 days (hazard ratio = 2.5; 95% CI, 0.9-7.3), and 31 of 493 (6.3%) when used for 3 days (hazard ratio = 3.8; 95% CI, 1.6-9.3; p = 0.002). Patients with any ciprofloxacin-containing antibiotic regimen the first 3 days in the trial had a higher risk of invasive Candida infection than did patients on any antibiotic regimen not containing ciprofloxacin (unadjusted hazard ratio = 3.7; 95% CI, 1.6-8.7; p = 0.003; adjusted hazard ratio, 3.4; 95% CI, 1.4-8.0; p = 0.006). CONCLUSIONS: High exposure to antibiotics is associated to increased risk of invasive Candida infection in medical intensive care patients. Patients with ciprofloxacin-containing regimens had higher risk of invasive Candida infection. Other antibiotics, such as meropenem, piperacillin/tazobactam, and cefuroxime, were not associated with such a risk.

Methods to improve adoptive T-cell therapy for melanoma: IFN-γ enhances anticancer responses of cell products for infusion.

Further development of adoptive T-cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has the potential to markedly change the long-term prognosis of patients with metastatic melanoma, and modifications of the original protocol that can improve its clinical efficacy are highly desirable. In this study, we demonstrated that a high in vitro tumor reactivity of infusion products was associated with clinical responses upon adoptive transfer. In addition, we systematically characterized the responses of a series of TIL products to relevant autologous short term-cultured melanoma cell lines from 12 patients. We provide evidence that antitumor reactivity of both CD8(+) and CD4(+) T cells could be enhanced in most TIL products by autologous melanoma sensitization by pretreatment with low-dose IFN-γ. IFN-γ selectively enhanced responses to tumor-associated antigens other than melanoma differentiation antigens. In addition, IFN-γ treatment was invariably associated with restored/increased cancer immunogenicity as demonstrated by upregulation of major histocompatibility complex molecules. These findings suggest a potential synergism between IFN-γ and ACT, and have important implications for clinical development of combination strategies for the treatment of metastatic melanoma.

Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma.

BACKGROUND: Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. PATIENTS AND METHODS: This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). RESULTS: Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. CONCLUSION: Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma.

Procalcitonin-guided interventions against infections to increase early appropriate antibiotics and improve survival in the intensive care unit: a randomized trial.

OBJECTIVE: For patients in intensive care units, sepsis is a common and potentially deadly complication and prompt initiation of appropriate antimicrobial therapy improves prognosis. The objective of this trial was to determine whether a strategy of antimicrobial spectrum escalation, guided by daily measurements of the biomarker procalcitonin, could reduce the time to appropriate therapy, thus improving survival. DESIGN: Randomized controlled open-label trial. SETTING: Nine multidisciplinary intensive care units across Denmark. PATIENTS: A total of 1,200 critically ill patients were included after meeting the following eligibility requirements: expected intensive care unit stay of ≥ 24 hrs, nonpregnant, judged to not be harmed by blood sampling, bilirubin <40 mg/dL, and triglycerides <1000 mg/dL (not suspensive). INTERVENTIONS: : Patients were randomized either to the "standard-of-care-only arm," receiving treatment according to the current international guidelines and blinded to procalcitonin levels, or to the "procalcitonin arm," in which current guidelines were supplemented with a drug-escalation algorithm and intensified diagnostics based on daily procalcitonin measurements. MEASUREMENTS AND MAIN RESULTS: The primary end point was death from any cause at day 28; this occurred for 31.5% (190 of 604) patients in the procalcitonin arm and for 32.0% (191 of 596) patients in the standard-of-care-only arm (absolute risk reduction, 0.6%; 95% confidence interval [CI] -4.7% to 5.9%). Length of stay in the intensive care unit was increased by one day (p = .004) in the procalcitonin arm, the rate of mechanical ventilation per day in the intensive care unit increased 4.9% (95% CI, 3.0-6.7%), and the relative risk of days with estimated glomerular filtration rate <60 mL/min/1.73 m was 1.21 (95% CI, 1.15-1.27). CONCLUSIONS: Procalcitonin-guided antimicrobial escalation in the intensive care unit did not improve survival and did lead to organ-related harm and prolonged admission to the intensive care unit. The procalcitonin strategy like the one used in this trial cannot be recommended.

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial.

BACKGROUND AND AIM: Dendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2+ patients) or with autologous/allogeneic tumor lysate (HLA-A2(−) patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-alpha2b. RESULTS: Of 46 patients who initiated treatment, 10 stopped treatment within 1-4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2+ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25(high) CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD. CONCLUSIONS: Vaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.

Identification and characterization of survivin-derived H-2Kb-restricted CTL epitopes.

Survivin is overexpressed in several malignancies and in tumor-associated endothelium making it an attractive target for therapeutic cytotoxic T-cell responses. Thus, it would be important to test this notion in preclinical models. Consequently, we screened the murine survivin sequence for potential binding K(b)-restricted octamer peptide epitopes. Two epitopes, which bind strongly to K(b), were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes. The natural processing and presentation of these epitopes by tumor cells was evident by the killing of murine melanoma cells by vaccination-induced T cells. Subcutaneous challenge with syngeneic melanoma demonstrated the protective immunity of this vaccination. Notably, analysis of the vessel density in subcutaneous tumors revealed that survivin-specific vaccination significantly reduced the number of intratumoral vessels. In summary, we demonstrated the immunogenicity of two K(b)-restricted peptide epitopes derived from the murine survivin protein; moreover, survivin-specific vaccination not only resulted in a reduction of tumor cells but also the tumor supplying blood vessels. The presented preclinical model for survivin-directed vaccination may serve as a valuable tool to improve already running clinical trials in a syngeneic tumor model.