Seasonal influenza outbreaks, especially in high-risk groups such as the elderly, represent an important public health problem. Prevailing inadequate efficacy of seasonal vaccines is a crucial bottleneck. Understanding the immunological and molecular mechanisms underpinning differential influenza vaccine responsiveness is essential to improve vaccination strategies. Here we show comprehensive characterization of the immune response of randomly selected elderly participants (≥ 65 years), immunized with the adjuvanted influenza vaccine Fluad. In-depth analyses by serology, multi-parametric flow cytometry, multiplex and transcriptome analysis, coupled to bioinformatics and mathematical modelling, reveal distinguishing immunological and molecular features between responders and non-responders defined by vaccine-induced seroconversion. Non-responders are specifically characterized by multiple suppressive immune mechanisms. The generated comprehensive high dimensional dataset enables the identification of putative mechanisms and nodes responsible for vaccine non-responsiveness independently of confounding age-related effects, with the potential to facilitate development of tailored vaccination strategies for the elderly.

Introduction/BackgroundAlmost all cervical cancers (CC) are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Studies has shown that most patients with HPV-associated tumors have detectable circulating HPV DNA (HPV ctDNA) in the blood at time of diagnosis, before treatment. Development in sensitive technologies led to the use of cell-free DNA for monitoring patients. In the present study, we investigated if HPV ctDNA may serve as a residual tumor marker at the end of chemo-radiation or to predict relapse during the follow-up period.MethodologyWe analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs (NCT02428842) prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse.ResultsCirculating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (p=0.02) and para-aortic lymph node involvement (p=0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R=0.39, p<0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (p<0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2–15) from HPV ctDNA detection.ConclusionHPV ctDNA detection is a useful marker to predict relapse in cervical cancer.

Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection. HPV ctDNA detection is a useful marker to predict relapse in cervical cancer.See related commentary by Wentzensen and Clarke, p. 5733.

BACKGROUNDPediatric AML represents a rare and heterogeneous disease. Within the BFM study group, HCT for this patient cohort was performed with considerable inter-center variability. AML SCT-BFM 2007 was based on a transplantation consensus and aimed to prospectively generate a reliable body of data on which further transplantation technologies could be based. METHODSAML SCT-BFM 2007 was conducted according to current legislation as an international multicenter clinical trial (EudraCT: 2007-004517-34, NCT00606723), data were collected via a specifically developed remote data entry (RDE)-based data bank, and verified against source data by an on-site monitoring program. Children with cytogentic and molecular high-risk features achieving a complete first remission (CR1) and those in CR2 after a first relapse qualified for HCT from a matched donor (MD). A MD was defined to be identical when at least 9/10 alleles on the HLA-A, B, C, DRB1 and DQ loci did match. A myeloablative conditioning regimen consisting of Busulfan (age adjusted i.v. dosing: 3.2 - 4,8 mg/kg BW on days -7 through -4), Cyclophosphamide (60mg/kg iv on days -3 and -2), and Melphalan (140 mg/m2 on day -1) termed BuCyMel was used. For GVHD prophylaxis CSA and short term MTX were given, for unrelated donors anti-thymocyte globulin (ATG Neovii) was added. Children with refractory primary disease or refractory relapse were eligible for early transplantation. A cytoreductive regimen containing Fludarabine (30mg/m2/d i.v.), Amsacrine (100 mg/m2/d i.v.) and Cytarabine (2g/m2/d i.v.) all on days -12 through -9 (FLAMSA) was immediately followed by a reduced intensity conditioning (RIC) consisting of 4 Gy TBI and Cyclophosphamide (60 (unrelated)/40 (related) mg/kg/d i.v.) on days -4 and -3. After early taper of immunosuppression (MMF halted by day 40 and CSA after taper by day 90) increasing doses of prophylactic donor lymphocyte infusions (DLI) were given on days 120, 150, and 180. Standard statistical methods were used for the analysis of event-free survival (EFS, events: relapse, death from any cause), overall survival (OS), cumulative incidence of treatment related mortality (TRM) and relapse (CIR) after HCT. RESULTSRecruitment was conducted between 5/2010 and 2/2016. 97 children underwent HCT after conditioning with BuCyMel. Median neutrophil engraftment was achieved after 21 days (range 9-38). 3-year EFS and OS were 62%, standard error (SE) 5%, and 73%, SE 5%. CIR was 22%, SE 4%. Median time to relapse was 8 months (range 1-66) after HCT. TRM was 15%, SE 4%, with 8/15 patients dying before day 100. TRM correlated with age with a rate of 9% in children younger than 12 years (n = 68) and 31% in older children and adolescents (Fig. A). This resulted in a stop of using BuCyMel for older children and adolescents in 6/2014. The rate of acute graft versus host disease (aGVHD) grade II-IV was 22%, chronic GVHD developed in 11%. Median neutrophil engraftment for children transplanted for refractory disease (n= 35) was 23 days (range 10-34). 3-year EFS and OS were 46%, SE 8%, and 51%, SE 9%. EFS for primarily refractory disease was 53%, SE 11%, whereas EFS for refractory relapses (n = 7) was 29%. Relapse rate was 43%, SE 9%, and TRM reached 11%, SE 5% (Fig. B). Acute and chronic GVHD rates were at 26% and 17%. CONCLUSIONWithin a well-defined trial concept HCT from a matched donor using BuCyMel for conditioning results in an EFS of 60% with a low TRM rate in younger children. The identical approach is associated with a high TRM rate of 31% in older children and adolescents. Whether the use of alternative conditioning strategies in this population can reduce mortality while maintaining the relapse-free survival rates remains to be determined. In high-risk patients with refractory disease, early use of RIC HCT followed by the infusion of prophylactically given DLI can induce long-term remissions in more than 50% of children without increasing the risk for GVHD. [Display omitted] DisclosuresSauer:Neovii: Research Funding. Lang:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Bader:Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding. Handgretinger:Miltenyibiotec GmbH: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.

BackgroundCOVID-19 prediction models based on clinical characteristics, routine biochemistry and imaging, have been developed, but little is known on proteomic markers reflecting the molecular pathophysiology of disease progression.MethodsThe multicentre (six European study sites) Prospective Validation of a Proteomic Urine Test for Early and Accurate Prognosis of Critical Course Complications in Patients with SARS-CoV-2 Infection Study (Crit-COV-U) is recruiting consecutive patients (≥ 18 years) with PCR-confirmed SARS-CoV-2 infection. A urinary proteomic biomarker (COV50) developed by capillary-electrophoresis-mass spectrometry (CE-MS) technology, comprising 50 sequenced peptides and identifying the parental proteins, was evaluated in 228 patients (derivation cohort) with replication in 99 patients (validation cohort). Death and progression along the World Health Organization (WHO) Clinical Progression Scale were assessed up to 21 days after the initial PCR test. Statistical methods included logistic regression, receiver operating curve (ROC) analysis and comparison of the area under the curve (AUC).FindingsIn the derivation cohort, 23 patients died, and 48 developed worse WHO scores. The odds ratios (OR) for death per 1 standard deviation (SD) increment in COV50 were 3·52 (95% CI, 2·02–6·13, p <0·0001) unadjusted and 2·73 (1·25–5·95, p = 0·012) adjusted for sex, age, baseline WHO score, body mass index (BMI) and comorbidities. For WHO scale progression, the corresponding OR were 2·63 (1·80–3·85, p<0·0001) and 3·38 (1·85–6·17, p<0·0001), respectively. The area under the curve (AUC) for COV50 as a continuously distributed variable was 0·80 (0·72–0·88) for mortality and 0·74 (0·66–0·81) for worsening WHO score. The optimised COV50 thresholds for mortality and worsening WHO score were 0·47 and 0·04 with sensitivity/specificity of 87·0 (74·6%) and 77·1 (63·9%), respectively. On top of covariates, COV50 improved the AUC, albeit borderline for death, from 0·78 to 0·82 (p = 0·11) and 0·84 (p = 0·052) for mortality and from 0·68 to 0·78 (p = 0·0097) and 0·75 (p = 0·021) for worsening WHO score. The validation cohort findings were confirmatory.InterpretationThis first CRIT-COV-U report proves the concept that urinary proteomic profiling generates biomarkers indicating adverse COVID-19 outcomes, even at an early disease stage, including WHO stages 1–3. These findings need to be consolidated in an upcoming final dataset.FundingThe German Federal Ministry of Health funded the study.

Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma1–3. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II4,5. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H)+ tumours in syngeneic MHC-humanized mice4,6–8. Here we describe a multicentre, single-arm, open-label, first-in-humans phase I trial that we carried out in 33 patients with newly diagnosed World Health Organization grade 3 and 4 IDH1(R132H)+ astrocytomas (Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), ClinicalTrials.gov identifier NCT02454634). The trial met its primary safety endpoint, with vaccine-related adverse events restricted to grade 1. Vaccine-induced immune responses were observed in 93.3% of patients across multiple MHC alleles. Three-year progression-free and death-free rates were 0.63 and 0.84, respectively. Patients with immune responses showed a two-year progression-free rate of 0.82. Two patients without an immune response showed tumour progression within two years of first diagnosis. A mutation-specificity score that incorporates the duration and level of vaccine-induced IDH1(R132H)-specific T cell responses was associated with intratumoral presentation of the IDH1(R132H) neoantigen in pre-treatment tumour tissue. There was a high frequency of pseudoprogression, which indicates intratumoral inflammatory reactions. Pseudoprogression was associated with increased vaccine-induced peripheral T cell responses. Combined single-cell RNA and T cell receptor sequencing showed that tumour-infiltrating CD40LG+ and CXCL13+ T helper cell clusters in a patient with pseudoprogression were dominated by a single IDH1(R132H)-reactive T cell receptor.

BACKGROUNDCervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality world wide and constitutes the third most common malignancy in women. The RAIDs consortium (http://www.raids-fp7.eu/) conducted a prospective European study [BioRAIDs (NCT02428842)] with the objective to stratify CC patients for innovative treatments. A “metagene” of genomic markers in the PI3K pathway and epigenetic regulators had been previously associated with poor outcome [2].METHODSTo detect new, more specific, targets for treatment of patients who resist standard chemo-radiation, a high-dimensional Cox model was applied to define dominant molecular variants, copy number variations, and reverse phase protein arrays (RPPA).FINDINGSSurvival analysis on 89 patients with all omics data available, suggested loss-of-function (LOF) or activating molecular alterations in nine genes to be candidate biomarkers for worse prognosis in patients treated by chemo-radiation while LOF of ATRX, MED13 as well as CASP8 were associated with better prognosis. When protein expression data by RPPA were factored in, the supposedly low molecular weight and nuclear form, of beta-catenin, phosphorylated in Ser552 (pβ-Cat552), ranked highest for good prognosis, while pβ-Cat675 was associated with worse prognosis.INTERPRETATIONThese findings call for molecularly targeted treatments involving p53, Wnt pathway, PI3K pathway, and epigenetic regulator genes. Pβ-Cat552 and pβ-Cat675 may be useful biomarkers to predict outcome to chemo-radiation, which targets the DNA repair axis.FUNDINGEuropean Union's Seventh Program for research, technological development and demonstration (agreement N°304,810), the Fondation ARC pour la recherche contre le cancer.

Acute graft-versus-host disease (aGvHD) contributes to about 50% of transplant-related mortality (non-relapse mortality) after allogeneic hematopoietic stem cell transplantation (HSCT). Here the predictive value of a urinary proteomic profile (aGvHD_MS17) was tested together with preemptive prednisolone therapy. Two-hundred and fifty-nine of 267 patients were eligible for analysis. Ninety-two patients were randomized upon aGvHD_MS17 classification factor above 0.1 to receive either prednisolone (2–2.5 mg/kg, N = 44) or placebo (N = 47; N = 1 randomization failure) for 5 days followed by tapering. The remaining 167 patients formed the observation group. The primary endpoint of the randomized trial was incidence of aGvHD grade II between randomization and day +100 post HSCT. Analysis of the short-term preemptive prednisolone therapy in the randomized patients showed no significant difference in incidence or severity of acute GvHD (HR: 1.69, 95% CI: 0.66–4.32, P = 0.27). Prednisolone as preemptive treatment did not lead to an increase in relapse (20.2% in the placebo and 14.0% in the prednisolone group (P = 0.46)). The frequency of adverse events was slightly higher in the placebo group (64.4% versus 50%, respectively). Taken together, the results of the Pre-GvHD trial demonstrated the feasibility and safety of preemptive prednisolone treatment in the randomized patients.

Doxorubicin is a standard of care in patients with advanced, inoperable soft tissue sarcoma (STS). We tested whether pazopanib has efficacy comparable to that of doxorubicin in elderly patients with STS and offers superior tolerability for hematologic toxicity. Patients age 60 years or older without previous systemic treatment for progressive advanced or metastatic STS who had Eastern Cooperative Oncology Group performance status of 0 to 2 and adequate organ function were included. Treatment consisted of pazopanib 800 mg once per day or doxorubicin 75 mg/m2 once every 3 weeks (≤ 6 cycles) after being randomly assigned in a 2:1 ratio. Noninferiority was assumed for progression-free survival (PFS), if the upper limit of the 95% CI for the hazard ratio (HR) was less than 1.8. Neutropenia and febrile neutropenia were key secondary end points. The European Organisation for Research and Treatment of Cancer (30-item) Quality of Life Questionnaire and geriatric assessment were used to measure patient-reported outcomes. Cox regression analysis and Kaplan-Meier curves were used for analysis. Pazopanib and doxorubicin were given to 81 and 39 patients, respectively. The median age was 71 years (range, 60-88 years). PFS was noninferior (HR, 1.00; 95% CI, 0.65 to 1.53) and the incidence of grade 4 neutropenia and febrile neutropenia favored pazopanib. Objective response rates for pazopanib and doxorubicin were 12.3% and 15.4%, respectively. Overall survival did not differ significantly between arms (HR, 1.08; 95% CI, 0.68 to 1.72; P = .735). Geriatric assessment revealed 2 or more comorbidities in 15.8% of the patients and impairment of activities of daily living in 28.3% of patients. Pazopanib was noninferior to doxorubicin, rendering pazopanib a putative therapeutic option in the first-line treatment of STS in patients age 60 years or older. The distinct adverse event profile may be used to counsel patients and tailor therapy to individual needs.

BackgroundThe standard therapy for patients suffering from sensorineural hearing loss is cochlear implantation. The insertion of the electrode array into the cochlea, with potential mechanical trauma and the presence of this foreign body inside the cochlea, may lead to free radical formation and reduced blood perfusion of the cochlea which can result in a loss of residual hearing. Studies have suggested that a particular combination of the antioxidants vitamins A, C and E as well as the vasodilator magnesium (together: ACEMg) may protect the residual hearing.MethodsThe potential protective effect of ACEMg on residual hearing preservation in cochlear implant (CI) patients was investigated in a single-centre, randomized, placebo-controlled, double-blind phase II clinical trial. CI candidates with some residual hearing in low frequencies receiving MED-EL implants of different FLEX electrode array lengths were treated with ACEMg tablets or placebo respectively 2 days preoperatively and up to 3 months postoperatively. The study objective was to demonstrate that ACEMg is more efficacious than placebo in preserving residual hearing during cochlear implantation by comparing the hearing loss (change in hearing thresholds at 500 Hz from baseline) 3 months after the first fitting between the two treatment groups and to investigate the treatments’ safety.ResultsFifty-one patients were included in the study, which had to be terminated before the recruitment goal was reached because of IMP-resupply mismanagement of one partner. In the intention-to-treat population, 25 patients were treated with ACEMg and 24 patients with placebo. The mean hearing loss at 500 Hz was (± 15.84) 30.21 dB (placebo) or (± 17.56) 26.00 dB (ACEMg) 3 months after the initial fitting. Adjusting the postoperative hearing loss for the baseline residual hearing, planned electrode length and surgeon results in 8.01 dB reduced hearing loss in ACEMg-treated patients compared to placebo-treated ones. The safety analysis revealed that ACEMg was generally well-tolerated with adverse event frequencies below the placebo level.ConclusionThis is the first clinical trial investigating a drug effect on residual hearing in CI patients. These first-in-man data may suggest that a perioperative oral administration of ACEMg is safe and may provide protection of residual hearing in CI patients.Trial registrationEU Clinical Trial Register No. 2012-005002-22. Registered on 6 December 2013. Funding: European Commission FP7-HEALTH-2012-INNOVATION-2.