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Background In patients with non-small cell lung cancer (NSCLC), accuracy of [18F]FDG-PET/CT for pretherapeutic lymph node (LN) staging is limited by false positive findings. Our aim was to evaluate machine learning with routinely obtainable variables to improve accuracy over standard visual image assessment. Methods Monocentric retrospective analysis of pretherapeutic [18F]FDG-PET/CT in 491 consecutive patients with NSCLC using an analog PET/CT scanner (training + test cohort, n = 385) or digital scanner (validation, n = 106). Forty clinical variables, tumor characteristics, and image variables (e.g., primary tumor and LN SUVmax and size) were collected. Different combinations of machine learning methods for feature selection and classification of N0/1 vs. N2/3 disease were compared. Ten-fold nested cross-validation was used to derive the mean area under the ROC curve of the ten test folds (“test AUC”) and AUC in the validation cohort. Reference standard was the final N stage from interdisciplinary consensus (histological results for N2/3 LNs in 96%). Results N2/3 disease was present in 190 patients (39%; training + test, 37%; validation, 46%; p = 0.09). A gradient boosting classifier (GBM) with 10 features was selected as the final model based on test AUC of 0.91 (95% confidence interval, 0.87–0.94). Validation AUC was 0.94 (0.89–0.98). At a target sensitivity of approx. 90%, test/validation accuracy of the GBM was 0.78/0.87. This was significantly higher than the accuracy based on “mediastinal LN uptake > mediastinum” (0.7/0.75; each p 10 mm; 0.68/0.75; each p Conclusions A machine learning model based on routinely available variables from [18F]FDG-PET/CT improved accuracy in mediastinal LN staging compared to established visual assessment criteria. A web application implementing this model was made available.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x(L), Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-x(L) and Mcl-1. Expression of Bcl-x(L), Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-x(L) and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatemnt with specific small molecule inhibitors of Bcl-x(L) (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-x(L) induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-x(L) and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-x(L) in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-x(L) as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Purpose To report on the clinical characteristics, outcome, and frequency of peritoneal carcinosis (PC) in patients with advanced germ cell tumors (GCT), a multicenter registry analysis was carried out. Methods A multicenter registry analysis was conducted by the German Testicular Cancer Study Group (GTCSG) with international collaborators. Data was collected and analyzed retrospectively. Patients were eligible for inclusion if PC was diagnosed either by radiologic or histopathologic finding during the course of disease. Descriptive and explorative statistical analysis was carried out with cancer-specific survival (CSS) as primary study endpoint. Results Collaborators from ten GCT expert centers identified 28 GCT (0.77%) patients with PC after screening approximately 3767 GCT patient files and one case was contributed from a cancer registry request. Patients were diagnosed from 1997 to 2019 at a median age of 37 years (interquartile range, 13). Two patients (7%) presented with stage I and 27 patients (93%) with synchronous metastatic disease at first diagnosis. The primary histology was seminoma in seven (27%) and non-seminoma in 21 patients (72%). PC was detected after a median of 15.3 months from primary diagnosis (range 0–177) and two consecutive treatment lines (range 0–5), respectively. The median CSS from the time of detection of PC was 10.5 months (95%Confidence Interval 0.47–1.30) associated with an overall 2-year CSS rate of 30%. Conclusion PC represents a rare tumor manifestation in GCT patients and was primarily associated with the occurrence of advanced cisplatin-refractory disease conferring to a dismal prognosis.

Introduction Pembrolizumab is a highly effective standard of care in PD-L1 overexpressing (≥ 50%) non–small-cell lung cancer. However, a substantial share of patients from everyday clinical practice is treated without clear evidence from clinical trials. Patients and Methods We performed a retrospective multicentric study including all consecutive patients from 6 certified lung cancer centers in Berlin, Germany, having received pembrolizumab as first-line palliative therapy from January 1 until December 31, 2017. Aims were to validate published clinical trials with a special focus on efficacy and outcome in patients with reduced performance status (PS), brain metastases, and steroids. Results A total of 153 patients were included (median age 69 years, 58% men, 69% adenocarcinoma). Rates for PS ≥ 2, brain metastases, and steroids were 24.8%, 20.9%, and 24.2%, respectively. Median objective response rate, progression-free and overall survival were 48.5%, 8.2 and 22.0 months for all patients and 52.4%, 8.8 and 29.2 months in patients fulfilling the inclusion criteria for the KEYNOTE-024 trial. Patients with a comorbidity-defined PS ≥ 2, symptomatic brain metastases requiring upfront radiotherapy, or baseline steroids had significantly reduced survival. In contrast, durable responses occurred with a tumor-related PS ≥ 2 or asymptomatic brain metastases. Grade 3/4 and 5 immune-related adverse events affected 13.7% and 2.0% of patients. Conclusion Real-world and clinical trial efficacy with upfront pembrolizumab correspond well. Pembrolizumab may sufficiently control asymptomatic brain metastases and may improve a cancer-related reduced PS. However, the frail share of patients with a comorbidity-defined PS ≥ 2, symptomatic brain metastases, or baseline steroids derives no relevant benefit.

Regional therapies for primary and secondary liver tumors have garnered interest in recent years and several types of treatment approaches have been pursued to control disease, palliate symptoms, and extend survival. Chemosaturation is an innovative way to deliver high-dose chemotherapy to the liver via the hepatic artery. Within the last decade, "isolated hepatic perfusion" (IHP) has evolved from an open surgical approach to a minimally invasive procedure, now termed "chemosaturation" (CS) with "percutaneous hepatic perfusion (PHP)". The most conclusive data on CS-PHP is currently available for patients with hepatic metastases from uveal melanoma (UM) - a rare but devastating disease with a poor long-term survival rate. A global phase-3 study and several cohort studies have provided compelling evidence that CS-PHP is an effective salvage treatment for liver-dominant metastatic UM in institutions with appropriate expertise. In this review we provide an overview on the technique, available clinical data, including safety and efficacy, and potential indications for CS-PHP.

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS- based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.Statement of significanceThe significance of this study lies not only in the potential of our optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) for clinical application, but sheds light on the entity-agnostic prevalence of HLA class I peptide presentation of RNA processing events to be used for tumor targeting.

Objectives In patients with NSCLC, current ESTS and ESMO guidelines recommend invasive lymph node (LN) staging with EBUS-TBNA even if FDG-PET/CT is negative for mediastinal LNs if at least one of three risk factors is present (cN1, non-peripheral primary or primary >3 cm). Modified workflows to avoid unnecessary invasive procedures were evaluated. Materials and methods Monocentric retrospective analysis of pretherapeutic FDG-PET/CT in 247 patients with NSCLC (62 % male; age, 68 [43–88] years) using an analog or digital PET/CT scanner. PET windowing was standardized. LNs were positive if ‘LN uptake > mediastinal blood pool’ or short axis >10 mm. Surgery or EBUS-TBNA served as reference for diagnostic accuracy per LN station. In all patients with negative mediastinal LNs by PET/CT, LN histology from surgery was available. Results Among 700 L N stations analyzed, 180 were malignant. Sensitivity and specificity of PET/CT per LN station were 93 % and 71 %. Following current guidelines, 76 patients with mediastinal negative PET/CT required confirmatory invasive staging. Only 5/76 patients had unexpected pN2 (all had adenocarcinoma). In a modified approach, confirmatory invasive staging was confined to patients with mediastinal negative PET/CT who showed all three risk factors. Using this modification, EBUS-TBNA could have been omitted in 62 (82 %) of the 76 patients who required EBUS-TBNA based on current recommendation. Among these 62 patients, only one patient had unsuspected pN2 (single-level) while the remaining 61 of 62 omitted EBUS-TBNA were deemed unnecessary because mediastinal LNs were confirmed to be negative. No multi-level pN2 would have been missed. Conclusion In the current analysis, 82 % of EBUS-TBNA procedures in patients with mediastinal negative PET/CT could have been omitted by modifying the current guideline workflow as proposed (i.e., restricting EBUS-TBNA in patients with cN0/1 to those with all three risk factors). This was consistent with different PET/CT scanners. Prospective confirmation is required.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x

4141 Background: Interleukin-1 Receptor Accessory Protein (IL1RAP) is expressed on cancer and stromal cells in PDAC. The IL-1 pathway is active in the pancreatic tumor microenvironment and upregulated in response to chemotherapy. IL1RAP interacts with IL-1R1 and modulates downstream factors (e.g. IL-6, IL-8) and CRP level. Nadunolimab (CAN04), a fully humanized ADCC-enhanced IgG1 antibody, targets IL1RAP and blocks IL-1α and IL-1β signaling. Here, results are reported from the phase 1/2a clinical trial CANFOUR evaluating nadunolimab combined with GN in PDAC. Methods: Patients (pts) with previously untreated, unresectable, locally advanced or metastatic PDAC received nadunolimab at 1 (n=20) 2.5 (n=20), 5 (n=28) or 7.5 mg/kg (n=8) Q2W with standard GN. Primary objective was safety; secondary objectives included ORR, iPFS per iRECIST and OS, and exploratory objectives included effects on serum and tumor tissue biomarkers. Results: In total, 76 pts were enrolled: median age 63 years (43-89), 42% female, 93% stage IV, 45% PS=0, 9% received adjuvant chemotherapy. Treatment-related adverse events (AE) of grade≥3 were reported in 72% with neutropenia being most frequent. G-CSF prophylaxis was useful in managing neutropenic events. The 7.5 mg/kg dose was above MTD due to neutropenia. Infusion-related reactions were reported in 29% (grade 3 in 3%). Peripheral sensory neuropathy was rare (only two grade 2 events). Common treatment-related grade 3/4 AE are shown below. Seventy-three pts received combination therapy and were included in the efficacy analysis. Three pts did not receive chemotherapy due to consent withdrawal (n=2) or clinical deterioration (n=1). Median iPFS was 7.3 months (95% CI 5.6-8.9) with 15 pts still on treatment, median OS was 13.2 months (10.0-19.1) with 62% of pts alive and 1-year survival of 59% (42-72%). ORR was 33% (22-45), disease control rate 63% (51-74%) and duration of response 5.7 months (3.9-11.1). ORR was similar for all dose levels. Low baseline CRP was favorable for OS; reduction of serum IL-8 and CRP during Cycle 1 correlated with positive impact on OS. Also, neutrophil-lymphocyte ratio was reduced throughout the trial, driven by the reduction in circulating neutrophils. IL1RAP expression on cancer and stromal cells was confirmed in tumor biopsies. Conclusions: Nadunolimab combined with GN shows promising iPFS and OS and manageable safety in PDAC pts. A phase 2/3 trial of nadunolimab combined with chemotherapy in PDAC is planned and nadunolimab is also currently evaluated in additional combination trials with chemotherapy or IO. Clinical trial information: NCT03267316. [Table: see text]

Background: VB N-01 is an open label phase 1/2a basket trial to evaluate safety, feasibility, and immunogenicity of a therapeutic DNA cancer vaccine VB10.NEO in patients with locally advanced or metastatic solid cancers. Each VB10.NEO vaccine contains up to 20 neoepitopes selected by the proprietary AI platform NeoSELECT and is designed to target antigen presenting cells using Nykode’ s modular vaccine platform known as Vaccibody™. Patients and Methods: The trial enrolled patients with locally advanced or metastatic solid cancers (renal cell carcinoma, urothelial cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, and melanoma), who did not obtain complete responses on immune checkpoint inhibitor therapy (CPI). Up to 14 VB10.NEO doses (3 mg per dose) were administered i.m. by PharmaJet Stratis® for up to 1 year in combination with standard of care (CPI and/or other anti-cancer therapies). Blood samples and tumor biopsies were collected at baseline and during treatment for evaluation of immune responses. Results: At cut-off of May 2022, 41 patients had received at least one vaccination with VB10.NEO. The vaccine was safe and well-tolerated with no new or additional toxicity reported beyond that expected for CPIs alone. All patients displayed immune responses to a minimum of 3 neoepitopes (average 53% of vaccine neoepitopes), assessed by in vitro stimulation (IVS) interferon-gamma ELISpot. T cell responses were elicited in both high and low tumor mutational burden patients (TMB range 2-69mut/Mb). Multiple vaccinations increased the breadth and magnitude of the immune responses with vaccine-induced T cell responses (de novo and/or amplified pre-existing) measured in 95% of eligible patients. IVS intracellular cytokine staining of selected patients demonstrated that the majority of neoepitopes induced polyfunctional CD8 T cells. TCR sequencing of baseline and on-treatment samples (6 patients) showed expansion of T cell clones in both blood and tumor with selected clones being expanded in both compartments. Conclusions: VB10.NEO was generally well tolerated in patients with various pre-treated and advanced cancers. Assessment of neoepitope-specific T cell reactivity demonstrated VB10.NEO-induced broad and long-lasting T cell responses, and the majority of tested neoepitopes activated polyfunctional CD8 T cells. Pre- and post-vaccination TCRseq analysis of blood and tumor samples demonstrated the presence of blood-expanded clones in the on-treatment tumor sample potentially indicating trafficking of VB10.NEO-expanded T cells to the tumor site. Citation Format: Jürgen Krauss, Angela Krakhardt, Stephan Eisenmann, Sebastian Ochsenreither, Kaja C.G Berg, Kushi Kushekhar, Lars-Egil Fallang, Mikkel W. Pedersen, Siri Torhaug, Karsten B. Slot, Karoline Schjetne. Individualized APC targeting VB10.NEO cancer vaccines induce broad neoepitope-specific CD8 T cell responses in patients with advanced or metastatic solid tumors: interim results from a phase 1/2a trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT274.

Background: Interleukin-1 Receptor Accessory Protein (IL1RAP) is expressed on cancer and stromal cells in pancreatic ductal adenocarcinoma (PDAC), and high tumor IL1RAP RNA expression is a negative prognostic marker1. Dimerization between IL1RAP and the IL-1 receptor is required for IL-1 signaling, and the IL-1 axis has been implicated in the tumor microenvironment and immune response to PDAC. Nadunolimab (CAN04), a fully humanized ADCC-enhanced IgG1 anti-IL1RAP antibody, blocks IL-1α and IL-1β signaling. Preliminary efficacy from the clinical phase 1/2a trial CANFOUR, where PDAC patients (n=73) received nadunolimab with gemcitabine and nab-paclitaxel (GN), show median iPFS of 7.2 months (iRECIST; 95% CI 5.2-8.5), median OS of 12.7 months (10.0-19.1) with 59% of patients alive, and 1-year survival of 57%2. Methods: Patients with previously untreated, locally advanced or metastatic PDAC received nadunolimab at doses from 1 to 7.5 mg/kg, in combination with GN. The exploratory objective was to measure serum and tumor biomarkers related to treatment outcome. Pre-treatment tumor biopsies were assessed for IL1RAP expression by immunohistochemistry. Serum from all patients was analyzed for biomarkers by Meso Scale Discovery (MSD) and Olink. Results: IL1RAP was expressed on tumor and stromal cells in all biopsies analysed and IL1RAP-positive infiltrating immune cells were detected in biopsies from 94% of patients (n=38). The level of IL1RAP on tumor cells was associated with response (PR vs SD and PD; p=0.082). Patients with PR had a median IL1RAP H-score of 200, while patients with SD or PD as best response had a median H-score of 110. The data suggest a relationship between high IL1RAP expression and increased iPFS and OS. Reductions in serum biomarkers downstream of IL-1, detected by MSD, were also associated with response. Patients with the largest treatment-related decreases in IL-6 displayed a prolonged iPFS (p Conclusions: Nadunolimab combined with GN shows promising iPFS and OS in 73 PDAC patients. In these patients, high tumor cell expression of IL1RAP, the molecular target for nadunolimab, correlated with response. Reduction of the downstream biomarkers IL-6 and IL-8, as well as CRP, were most pronounced in responding patients and correlated with efficacy. These data strongly suggest that nadunolimab interaction with IL1RAP is important for the treatment effects observed in the CANFOUR study. 1TCGA Database2Van Cutsem et al; ASCO 2022 Citation Format: Eric Van Cutsem, Joelle Collignon, Rikke Eefsen, Sebastian Ochsenreither, Zanete Zvirbule, Audrius Ivanauskas, Camilla Rydberg Millrud, Petter Skoog, Nedjad Losic, Susanne Magnusson, Annika Sanfridson, David Liberg. Tumor IL1RAP levels and reduction in serum biomarkers correlate with response in PDAC patients treated with nadunolimab, an anti-IL1RAP monoclonal antibody, in combination with gemcitabine and nab-paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2172.

Thyroid transcription factor 1 (TTF-1) is a prognostic biomarker in lung adenocarcinoma; however, TTF-1-positive patients also display more favorable factors like actionable target mutations. In contrast, TTF-1-negative cancer is a poorly described entity. We performed a retrospective study to characterize a TTF-1-negative phenotype and to evaluate outcome depending on the chemotherapy regimen applied in the EGFR/ALK-negative population.Phenotypic traits were analyzed in 741 patients with evaluable TTF-1 expression status, among them 529 patients with platinum-based first-line chemotherapy, with disease diagnosed between 2009 and 2016 at a tertiary referral university hospital. The influence of TTF-1 and several cofactors on progression-free survival and overall survival (OS) were analyzed using a 1:1 propensity score matching model, depending on the platinum doublet chemotherapy's incorporating pemetrexed or not, with subsequent Cox regression.TTF-1 negativity implied a distinct cancer phenotype with the predominance of male sex, worse Eastern Cooperative Oncology Group performance status, greater metastatic burden at primary diagnosis, and more adrenal gland metastases. These patients had improved progression-free survival (hazard ratio, 0.42; P = .001) and OS (hazard ratio, 0.40; P .001) when gemcitabine-, taxane-, or vinorelbine-based regimens were provided instead of pemetrexed. None of the regimens was superior in TTF-1-positive patients with regard to OS. Overall, TTF-1 expression was strongly prognostic with a substantial increase in progression-free survival (hazard ratio, 0.54; P .001) and OS (hazard ratio, 0.53; P .001).TTF-1 negativity is associated with a distinct cancer phenotype. Incorporation of this biomarker may be helpful when choosing an appropriate therapy regimen.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410–420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.

Targeted therapies for metastatic uveal melanoma have shown limited benefit in biomarker-unselected populations. The Treat20 Plus study prospectively evaluated the feasibility of a precision oncology strategy in routine clinical practice.Fresh biopsies were analyzed by high-throughput genomics (whole-genome, whole-exome, and RNA sequencing). A multidisciplinary molecular and immunologic tumor board (MiTB) made individualized treatment recommendations based on identified molecular aberrations, patient situation, drug, and clinical trial availability. Therapy selection was at the discretion of the treating physician. The primary endpoint was the feasibility of the precision oncology clinical program.Molecular analyses were available for 39/45 patients (87%). The MiTB provided treatment recommendations for 40/45 patients (89%), of whom 27/45 (60%) received ≥1 matched therapy. First-line matched therapies included MEK inhibitors (n = 15), MET inhibitors (n = 10), sorafenib (n = 1), and nivolumab (n = 1). The best response to first-line matched therapy was partial response in one patient (nivolumab based on tumor mutational burden), mixed response in two patients, and stable disease in 12 patients for a clinical benefit of 56%. The matched therapy population had a median progression-free survival and overall survival of 3.3 and 13.9 months, respectively. The growth modulation index with matched therapy was1.33 in 6/17 patients (35%) with prior systemic therapy, suggesting clinical benefit.A precision oncology approach was feasible for patients with metastatic uveal melanoma, with 60% receiving a therapy matched to identify molecular aberrations. The clinical benefit after checkpoint inhibitors highlights the value of tumor mutational burden testing.

Background Approvals of cancer therapeutics are primarily disease entity specific. Current molecular diagnostic approaches frequently identify actionable alterations in rare cancers or rare subtypes of common cancers for which the corresponding treatments are not approved and unavailable within clinical trials due to entity-related eligibility criteria. Access may be negotiated with health insurances. However, approval rates vary, and critical information required for a scientific evaluation of treatment-associated risks and benefits is not systematically collected. Thus clinical trials with optimized patient selection and comprehensive molecular characterization are essential for translating experimental treatments into standard care. Patients and methods Continuous ReAssessment with Flexible ExTension in Rare Malignancies (CRAFT) is an open-label phase II trial for adults with pretreated, locally advanced, or metastatic solid tumors. Based on the evaluation by a molecular tumor board, patients are assigned to combinations of six molecularly targeted agents and a programmed death-ligand 1 (PD-L1) antagonist within seven study arms focusing on (i) BRAF V600 mutations; (ii) ERBB2 amplification and/or overexpression, activating ERBB2 mutations; (iii) ALK rearrangements, activating ALK mutations; (iv and v) activating PIK3CA and AKT mutations, other aberrations predicting increased PI3K–AKT pathway activity; (vi) aberrations predicting increased RAF–MEK–ERK pathway activity; (vii) high tumor mutational burden and other alterations predicting sensitivity to PD-L1 inhibition. The primary endpoint is the disease control rate (DCR) at week 16; secondary and exploratory endpoints include the progression-free survival ratio, overall survival, and patient-reported outcomes. Using Simon’s optimal two-stage design, 14 patients are accrued for each study arm. If three or fewer patients achieve disease control, the study arm is stopped. Otherwise, 11 additional patients are accrued. If the DCR exceeds 7 of 25 patients, the null hypothesis is rejected for the respective study arm. Conclusions CRAFT was activated in October 2021 and will recruit at 10 centers in Germany. Trial registration numbers EudraCT: 2019-003192-18; ClinicalTrials.gov: NCT04551521., Highlights • Actionable genetic alterations are detected in many cancers with unknown efficacy of the corresponding targeted therapies. • CRAFT, an open-label multicenter phase II trial, uses six molecularly targeted agents and a PD-L1 antagonist in seven treatment arms. • Patient allocation to trial arms is based on evaluation of molecular tumor characteristics by the German Cancer Consortium. • The in-depth molecular characterization helps to understand mechanisms underlying primary and acquired therapy resistance. • The CRAFT trial has been active in Germany since October 2021 and will open at 10 sites during 2022.

9511 Background: The liver is the most common site of metastases in patients (pts) with uveal melanoma (UM). Here, we present results from the prespecified first interim analysis of the SirTac trial, a prospective, single-center, randomized, investigator-initiated, open-label phase 2 study of Selective Internal Radiation Therapy with Yttrium-90-bearing resin microspheres; SIR-Spheres (SIRT) vs Transarterial Chemoembolization with Cisplatin and degradable starch microspheres; EmboCept S (DSM-TACE) in pts with liver-dominant metastatic UM. Methods: Pts with histologically confirmed, liver-dominant metastatic UM and ECOG PS 0-2 were enrolled and randomized 1:1 to SIRT or DSM-TACE. Randomization was stratified by lactate dehydrogenase (

e21598 Background: Metastatic uveal melanoma (mUM) historically has a poor prognosis where chemotherapy and checkpoint blockade, including combination, has had no impact on OS. Tebentafusp (tebe), a TCR bispecific (gp100 x CD3), is the first therapy to demonstrate an OS benefit in a mUM randomized trial (IMCgp100-202) against pembrolizumab (pembro), ipilimumab (ipi) or dacarbazine. Here we present a summary of OS from recent prospective trials in mUM in both previously untreated (1L) and previously treated (2L+) mUM including cross trial comparison with checkpoint blockade combination. Methods: A literature review of prospective Ph2/3 clinical trials of systemic therapies in mUM published recently (2019-2021) identified 8 trials: in 1L mUM (n = 2) a randomized Ph3 of tebe (N = 252) vs investigator’s choice (IC; N = 126) of pembro, ipi or dacarbazine (IMCgp100-202) and a single arm Ph2 of nivolumab (nivo) + ipi (N = 52; Piulats, 2021) (Table); in 2L+ mUM (n = 1) a single arm Ph2 of tebe (N = 127; IMCgp100-102); and in mixed line mUM (n = 5) a randomized Ph2 of cabozantinib (N = 31) vs chemotherapy (N = 15; Luke, 2019), a single arm Ph2 of nivo + ipi (N = 35; Pelster, 2021), a single arm Ph2 of pembro + entinostat (N = 29; Ny, 2021), a single arm Ph2 of glembatumumab (N = 37; Hasanov, 2020), and a single arm Ph2 of IMC-A12 (IGF-1 receptor inhibitor; N = 18; Mattei, 2020). The PUMMA (Khoja, 2019; N = 912) and Rantala, 2019 (N = 2494; n = 510 1L pts) meta-analyses were included to provide historical benchmarks for OS. Results: In previously untreated mUM, 1-yr and median OS were 73% and 21.7 months for tebe, 59% and 15.7 months for pembro, 52% and 12.7 months for nivo + ipi and 45% and 11.3 months for conventional chemotherapy. In previously treated mUM, 1-yr and median OS were 61% and 16.8 months for tebe, 34% and 7.0 months for checkpoint inhibitors, and 43% and 10.5 months for conventional chemotherapy. In mixed line (1L+) mUM, 1-yr and median OS were 56% and 19.1 months for nivo + ipi and 59% and 13.4 months for pembro + entinostat, 13.8 months for IMC-A12, 11.9 months for glembatumumab and 6.4 months for cabozantinib. Related AEs (any grade) leading to discontinuation and death for tebe were 2% and 0% vs 5% and 0% for IC control, and 23% and 3.8% for nivo + ipi in 1L pts. Conclusions: Tebe is the only therapy demonstrated to prolong OS in mUM. The 1-yr OS for tebe in 1L and 2L+ is superior to all recent published studies, including nivo + ipi. Both tebe and checkpoint combination therapy have high rate of any grade AEs, although checkpoint combination has a higher rate of treatment related discontinuations and deaths.[Table: see text]

Background The aim of the RESGEX study was to compare the efficacy and safety of the anti-epidermal growth factor receptor (anti-EGFR) antibody tomuzotuximab against cetuximab both in combination with chemotherapy in patients with recurrent and/or metastatic squamous cell cancer of the head and neck in the first-line treatment. Patients and methods In this phase II trial 240 patients were equally randomized for six cycles to receive either tomuzotuximab (initial dose 990 mg then 720 mg) weekly and cisplatin 100 mg/m2 and fluorouracil (5-FU; 1000 mg/m2/day, days 1-4) every 3 weeks or cetuximab (400 mg/m2 subsequent 250 mg/m2) weekly with the same chemotherapeutic backbone followed by antibody maintenance treatment. The primary endpoint was progression-free survival. Results Median progression-free survival was 6.5 months [95% confidence interval (CI) 5.9-7.9 months] in the tomuzotuximab group and 6.2 months (95% CI 5.8-7.3 months) in the cetuximab group (P = 0.86). The median overall survival (OS) estimate was 11.6 months (95% CI 9.5-17.2 months) in the tomuzotuximab group and 13.8 months (95% CI 12.3-16.4 months) in the cetuximab group (P = 0.96). In an exploratory analysis a small subgroup of p16-positive patients had a significantly longer OS compared with p16-negative patients (hazard ratio 1.860, 95% CI 1.09-3.16, P = 0.02). Conclusions The glyco-engineered antibody tomuzotuximab failed to demonstrate improved efficacy with a chemotherapeutic backbone in the first-line treatment of recurrent or metastatic head and neck squamous cell carcinoma. It remains a so far unanswered question whether such antibody would partner better with different drugs such as checkpoint inhibitors., Highlights • Tomuzotuximab has a potential higher antibody-dependent cell cytotoxicity than other EGFR-directed antibodies. • Comparison of two anti-EGFR antibodies combined with chemotherapy in patients with squamous cell cancer of head and neck. • Efficacy, safety, and tolerability of tomuzotuximab and cetuximab in combination with chemotherapy were similar.

BACKGROUND: Pembrolizumab is a standard of care as first line palliative therapy in PD-L1 overexpressing (≥50%) non-small cell lung cancer (NSCLC). This study aimed at the identification of KRAS and TP53-defined mutational subgroups in the PD-L1 high population to distinguish long-term responders from those with limited benefit. METHODS: In this retrospective, observational study, patients from 4 certified lung cancer centers in Berlin, Germany, having received pembrolizumab monotherapy as first line palliative treatment for lung adenocarcinoma (LuAD) from 2017 to 2018, with PD-L1 expression status and targeted NGS data available, were evaluated. RESULTS: A total of 119 patients were included. Rates for KRAS, TP53 and combined mutations were 52.1%, 47.1% and 21.9%, respectively, with no association given between KRAS and TP53 mutations (P=0.24). By trend, PD-L1 expression was higher in KRAS-positive patients (75% vs. 65%, P=0.13). Objective response rate (ORR), median progression-free survival (PFS) and overall survival (OS) in the KRAS(G12C) group (n=32, 51.6%) were 63.3%, 19.8 months (mo.) and not estimable (NE), respectively. Results in KRAS(other) and wild type patients were similar and by far lower (42.7%, P=0.06; 6.2 mo., P

Background/Aim: High risk Human papillomavirus (hr-HPV) and smoking are independant risk factors for head and neck squamous cell carcinomas (HNSCC). While hr-HPV(+) HNSCC has a better prognosis than smoking-associated HNSCC no systematic data are yet available about the combined risk. Patients and Methods: We performed a meta-analysis to assess the overall survival of HNSCC patients relative to the hr-HPV and smoking status. A literature review up to November 2019 was conducted in PubMed and Cochrane Library using the search terms ‘HPV, Smoking and HNSCC’. Results: Nine out of 748 articles were included, 1,436 out of 2,080 patients were hr-HPV(+). The prevalence of hr-HPV(+) smokers was 36%. The meta-analysis showed a significantly better 5-year overall survival for HPV(+) non-smokers compared to smokers with risk ratio of 1.94 (95% confidence intervaI=1.46-2.58). Conclusion: Smoking is a negative prognostic factor for overall survival in patients with hr-HPV(+) HNSCC and should thus be an important part of staging and treatment.

The poor prognosis of locally advanced and metastatic head and neck squamous cell carcinoma (HNSCC) is primarily mediated by the functional properties of cancer stem cells (CSCs) and resistance to chemoradiotherapy. We investigated whether the aldehyde dehydrogenase (ALDH) inhibitor disulfiram (DSF) can enhance the sensitivity of therapy. Cell viability was assessed by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and apoptosis assays, and the cell cycle and reactive oxygen species (ROS) levels were evaluated by fluorescence-activated cell sorting (FACS). The radio-sensitizing effect was measured by a colony formation assay. The synergistic effects were calculated by combination index (CI) analyses. The DSF and DSF/Cu2+ inhibited the cell proliferation (inhibitory concentration 50 (IC50) of DSF and DSF/Cu2+ were 13.96 μM and 0.24 μM). DSF and cisplatin displayed a synergistic effect (CI values were

OBJECTIVE In the 8th Edition TNM Classification for Head and Neck Cancer, the classification for carcinoma of unknown primary (CUP) changed in addition to oropharyngeal carcinomas. The current classification considers extranodal extension (ENE), determination of p16 (surrogate marker for human papillomavirus), and detection of Epstein-Barr virus (EBV). The aim of this study was to investigate the influence of the new classification on the prognosis of p16-positive and p16-negative CUP and the impact of EBV proof. METHODS Clinical and pathological data from patients with CUP of the head and neck between 2009 and 2018 were evaluated. The 7th (UICC7) and 8th (UICC8) edition of the Union for International Cancer Control staging system were applied and compared. RESULTS There were 97 patients treated, 26.8% women and 73.2% men. The average age at initial diagnosis was 64.6 years. Of which, 58.8% had a documented history of smoking, 37.1% were positive for p16, 4.1% were positive for EBV, and 66% had ENE. Most of the patients were at stage III/IVa (78.4% according to UICC7). According to UICC8, p16+ patients were mainly at stage I (86.1%), and p16- at stage IVb (56.1%). P16 status (P = .002), ENE (P = .001), nodal category (TNM7, P

Background CV8102 is a non-coding, non-capped RNA complexed with a carrier peptide activating the innate (via TLR7/8, RIG-I) and adaptive immune system.1 2 An ongoing phase I trial is investigating i.t. CV8102 either as a single agent or in combination with systemic anti-PD-1 antibodies in patients with advanced melanoma (MEL), squamous cell carcinoma of the skin (cSCC) or head and neck (hnSCC) and adenoid cystic carcinoma (ACC). Methods An open-label, cohort-based, dose escalation and expansion study in patients with advanced cutaneous melanoma (cMEL), cutaneous squamous cell carcinoma (cSCC), head and neck squamous cell carcinoma (hnSCC) or adenoid cystic carcinoma (ACC) is ongoing investigating i.t. CV8102 as single agent and in combination with anti-PD-1 antibodies. 8 intratumoral injections of CV8102 are being administered initially over a 12 week period, while patients benefiting from the single agent therapy may receive further treatment. In an initial dose escalation part the maximum tolerated dose and recommended phase 2 dose for subsequent cohort expansion will be defined. Results As of September 16, 2020, 29 patients have been treated with CV8102 as a single agent (25-900 µg) and 21 patients have received CV8102 (25-900 µg) in combination with anti-PD-1 antibodies. Most frequent treatment related adverse events were mild to moderate fever, fatigue, chills and headache. One patient treated at the 900 µg single agent experienced a dose limiting toxicity (G3 transaminase increase in the context of G2 cytokine release syndrome). Regression of injected and distant noninjected lesions was observed in several patients in the single agent and the anti-PD-1 combination cohorts. Updated safety and efficacy results will be presented. Conclusions CV8102 showed an acceptable tolerability and preliminary evidence of clinical efficacy as single agent and in combination with anti-PD-1- antibodies. Trial Registration NCT03291002 Ethics Approval The study was approved by the Central Ethics Committees in Tuebingen, Germany under 785/2016AMG1, in France under 19.05.17.64111, in Barcelona, Spain under the EudraCT number. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. References Ziegler A, Soldner C, Lienenklaus S, Spanier J, Trittel S, Riese P, Kramps T, Weiss S, Heidenreich R, Jasny E, Guzman CA, Kallen KJ, Fotin-Mleczek M, Kalinke U. A new RNA-based adjuvant enhances virus-specific vaccine responses by locally triggering TLR- and RLH-dependent effects. J Immunol 2017;198(4):1595-1605. doi:10.4049/jimmunol.1601129 Heidenreich R, Jasny E, Kowalczyk A, Lutz J, Probst J, Baumhof P, Scheel B, Voss S, Kallen KJ, Fotin-Mleczek M. A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile. Int J Cancer 2015 Jul 15;137(2):372-84. doi: 10.1002/ijc.29402

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.

BackgroundCV8102 is a non-coding, non-capped RNA complexed with a carrier peptide activating the innate (via TLR7/8, RIG-I) and adaptive immune system.1 2 An ongoing phase I trial is investigating the intratumoral (i.t.) administration of CV8102 in patients with advanced cutaneous melanoma (cMEL), squamous cell carcinoma of the skin (cSCC) or head and neck (hnSCC) and adenoid cystic carcinoma (ACC), either as a single agent or in combination with systemic anti-PD-1 antibodies. Preliminary immune profiling results will be reported.MethodsAn open-label, cohort-based, dose escalation and expansion study in patients with advanced cMEL, cSCC, hnSCC or ACC is ongoing investigating CV8102 i.t. as single agent and in combination with anti-PD-1 antibodies. Eight i.t. injections of CV8102 were administered over a 12 week period with optional continuing treatment in case of clinical benefit. In the initial dose escalation part, the recommended phase II dose for subsequent cohort expansion was defined. Blood samples for immune cell phenotyping, RNA sequencing (RNAseq) and serum cytokine/chemokine analysis were collected at baseline and multiple time points during the treatment period. For characterization of the tumor microenvironment (TME), optional core needle biopsies of injected and/or non-injected lesions were taken before, during and after treatment. Changes on various tumor-infiltrating immune cells were assessed by multiplex immunofluorescence (MultiOmyx < sup >TM ) and immune-related gene expression profiling using nCounter® Pan Cancer IO360 < sup >TM panel (NanoString).ResultsDuring the dose escalation part, 33 patients received CV8102 (dose range of 25–900 µg) as single agent and 25 patients received CV8102 in combination with an anti-PD-1 antibody. A dose of 600 µg was selected as recommended phase II dose. Serum cytokine/chemokine and blood RNAseq analysis showed transient increases in several markers like interferons alpha and gamma after the first dose. First analyses of paired biopsies showed changes in the TME of injected and non-injected lesions. Complete results of cytokine and chemokine analysis in serum and blood RNAseq for the dose escalation cohorts will be presented. Multiplex immunofluorescence and gene expression profiling from paired biopsies from individual patients will be also included.ConclusionsIntratumoral injection of CV8102 activated several cytokine/chemokine pathways in the peripheral blood and showed immunological changes in the tumor microenvironment of injected and non-injected lesions.Trial RegistrationNCT03291002ReferencesZiegler A, Soldner C, Lienenklaus S, Spanier J, Trittel S, Riese P, Kramps T, Weiss S, Heidenreich R, Jasny E, Guzmán CA, Kallen KJ, Fotin-Mleczek M, Kalinke U. A New RNA-Based Adjuvant Enhances Virus-Specific Vaccine Responses by Locally Triggering TLR- and RLH-Dependent Effects. J Immunol 2017;198(4):1595–1605. doi: 10.4049/jimmunol.1601129.Heidenreich R, Jasny E, Kowalczyk A, Lutz J, Probst J, Baumhof P, Scheel B, Voss S, Kallen KJ, Fotin-Mleczek M. A novel RNA-based adjuvant combines strong immunostimulatory capacities with a favorable safety profile. Int J Cancer 2015 Jul 15;137(2):372–84. doi: 10.1002/ijc.29402.Ethics ApprovalThe study was approved by the Central Ethics Committees in Tuebingen, Germany under 785/2016AMG1, in France by the COMITE DE PROTECTION DES PERSONNES SUD-EST I under 2019–49, approval dated 17-May-2019, in Barcelona, Spain by the CEC COMITÉ DE ÉTICA DE INVESTIGACIÓN CLÍNICA CON MEDICAMENTOS del Hospital Universitari Vall d’Hebron, approval date 28-Nov-2019 under the EUdraCT number, in Austria by the Central Ethics Committee in Graz under 31–426 ex 18/19 approved on 19-Sep-2019.ConsentWritten informed consent from the patient was obtained for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Background: Metastatic uveal melanoma (mUM) has a poor prognosis with a 1-yr OS rate of 52%. No systemic treatment has proven an OS benefit in randomized trials. Tebentafusp (tebe), a bispecific consisting of an affinity-enhanced T cell receptor (TCR) fused to an anti-CD3 effector that can redirect T cells to target gp100+ cells, has shown promising activity in previously treated mUM pts. Here, we report the primary analysis of overall survival (OS) in the intention-to-treat population (ITT) of a Ph3 trial of tebe vs. investigator's choice (IC) as first line (1L) therapy in pts with mUM [NCT03070392]. Materials and Methods: In this randomized, open-label, Ph3 trial, 1L HLA-A*02:01+ pts with mUM were randomized 2:1 to receive tebe or IC of pembrolizumab, ipilimumab or dacarbazine, stratified by LDH. The primary endpoint was OS, defined as the time from randomization to death from any cause. Dual primary objectives were to evaluate 1) OS in the ITT population by comparing all tebe-randomized pts to all IC-randomized pts; and 2) OS in tebentafusp-treated patients with rash during week 1 versus all IC-treated patients. Secondary endpoints included safety and RECIST-defined overall response rate (ORR), progression free survival (PFS) and disease control rate (DCR). Here we present the OS in the ITT population. The study was unblinded by an independent data monitoring committee at the first pre-specified interim analysis. Investigator-reported radiographic-based endpoints were not mature at the first interim analysis. This analysis was conducted on the first interim analysis (data extracted Nov 2020). Results: 378 pts were randomized to tebe (252) or IC, including pembrolizumab (103), ipilimumab (15) or dacarbazine (7). Tebe significantly prolonged OS compared to IC (HR 0.51; 95% CI 0.36-0.71; PULN and versus pembrolizumab IC. Most common TRAEs were skin-related (gp100+ melanocytes) or cytokine-mediated (T cell activation) and included pyrexia, pruritus, and rash. These AEs decreased in frequency and severity after the first 3-4 doses and were generally manageable with standard interventions. In the tebe arm, the rate of treatment discontinuation due to TRAEs was low ( Conclusions: In 1L treatment of mUM pts, tebe monotherapy significantly improved OS compared to IC; the first investigational therapy to improve OS in pts with mUM. Tebe had a predictable and manageable AE profile with a low rate of related discontinuation. Tebe is the first TCR therapeutic to demonstrate an OS benefit. Citation Format: Sophie Piperno-Neumann, Jessica C. Hassel, Piotr Rutkowski, Jean-Francois Baurain, Marcus O. Butler, Max Schlaak, Ryan J. Sullivan, Sebastian Ochsenreither, Reinhard Dummer, John M. Kirkwood, Anthony M. Joshua, Joseph J. Sacco, Alexander N. Shoushtari, Marlana Orloff, Richard D. Carvajal, Omid Hamid, Shaad E. Abdullah, Chris Holland, Howard Goodall, Paul Nathan. Phase 3 randomized trial comparing tebentafusp with investigator's choice in first line metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT002.

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8

Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3β. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRβ chain libraries pre-enriched for target antigen specificity. Several TCRβ chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

Immunologische Therapieformen wie die Gabe von Antikorpern kommen bei soliden Tumoren, wie dem Plattenepithelkarzinom des Kopf-Hals-Bereichs entweder alleine, oder kombiniert mit Radio- oder Chemotherapie zum Einsatz. Trotz einiger respektabler Erfolge stost auch diese Therapieform an ihre Grenzen, nicht zuletzt aufgrund verschiedener Fahigkeiten des Tumors, sich dem Selektionsdruck des Immunsystems zu entziehen. Eine wesentliche Rolle scheinen hier Tumorstammzellen zu spielen, welche eine intrinsische Resistenz gegen konventionelle Therapien aufweisen und die Fahigkeit besitzen, die Heterogenitat der Tumormasse zu rekonstruieren. Auf diese Weise haben sie substantiellen Anteil an der Entwicklung von Rezidiven und Metastasen. Entsprechend sollten kunftige Immuntherapien auf diese Subpopulation spezifisch abzielen, moglicherweise auch in Kombination mit anderen Therapieformen. In dieser Ubersichtsarbeit werden die immunologischen Merkmale von Tumorstammzellen und ihr Potenzial als Ansatzpunkte fur eine Immuntherapie zusammengefasst.

Background Reliable and reproducible interpretation of molecular aberrations constitutes a bottleneck of precision medicine. Evidence-based decision management systems may improve rational therapy recommendations. To cope with an increasing amount of complex molecular data in the clinical care of patients with cancer, we established a workflow for the interpretation of molecular analyses. Methods A specialized physician screened results from molecular analyses for potential biomarkers, irrespective of the diagnostic modality. Best available evidence was retrieved and categorized through establishment of an in-house database and interrogation of publicly available databases. Annotated biomarkers were ranked using predefined evidence levels and subsequently discussed at a molecular tumour board (MTB), which generated treatment recommendations. Subsequent translation into patient treatment and clinical outcomes were followed up. Results One hundred patients were discussed in the MTB between January 2016 and May 2017. Molecular data were obtained for 70 of 100 patients (50 whole exome/RNA sequencing, 18 panel sequencing, 2 immunohistochemistry (IHC)/microsatellite instability analysis). The MTB generated a median of two treatment recommendations each for 63 patients. Thirty-nine patients were treated: 6 partial responses and 12 stable diseases were achieved as best responses. Genetic counselling for germline events was recommended for seven patients. Conclusion The development of an evidence-based workflow allowed for the clinical interpretation of complex molecular data and facilitated the translation of personalized treatment strategies into routine clinical care. The high number of treatment recommendations in patients with comprehensive genomic data and promising responses in patients treated with combination therapy warrant larger clinical studies.

Outcomes of multiply relapsed, refractory germ-cell tumour (GCT) patients remain poor with an overall survival (OS) of a few months only. Thus, new therapeutic advances are urgently needed. Cabazitaxel has shown preclinical activity in platinum-resistant GCT models. Here, we report the first clinical case series of cabazitaxel treatment for platinum-refractory GCT. Data of multiply relapsed GCT patients receiving single-agent cabazitaxel were retrospectively analysed. Endpoints included 12-week progression-free survival (PFS) rate, disease control rate, tumour marker responses, median PFS and OS, and toxicity. Cabazitaxel showed limited activity in 13 heavily pre-treated GCT patients. After a median follow-up of 23 weeks (IQR 29), 69% of patients were deceased. A median of 2 cycles of cabazitaxel (range 1–7) were applied. The 12-week PFS rate was 31%. Median PFS and OS were 7 and 23 weeks, respectively. Two patients achieved objective responses (15%), three patients (23%) achieved a tumour marker decline ≥ 50%, and the disease control rate was 39%. Cabazitaxel was well tolerated. CTCAE° III–IV haemato-toxicity was most common (54%), and dose reductions were scarce (15%). In this case series, cabazitaxel showed limited activity in heavily pre-treated GCT patients. Two-phase II studies are underway (NCT02115165, NCT02478502) prospectively assessing cabazitaxel in multiply relapsed GCTs.

In patients with non-small-cell lung cancer (NSCLC), asphericity (ASP) of the primary tumor's metabolic tumor volume (MTV) has shown prognostic significance. This study aimed at validation in an independent and sufficiently large cohort.A retrospective study was performed of 311 NSCLC patients undergoingEvents (progression and relapse) occurred in 167 of 311 patients; 137 died (median survivor follow-up, 37 months). In multivariable Cox regression for OS, ASP33.3% (hazard ratio, 1.58 [1.04-2.39]), male sex (1.84), age (1.04 per year), Eastern Cooperative Oncology Group performance status ≥ 2 versus 0/1 (2.68), stage II versus I (1.96), and Rx/1 versus R0 resection (2.1) were significant. Among separate UICC stages, ASP only predicted OS in stage II (optimal,19.5%; median OS, 33 vs. 59 months). Regarding PFS, ASP21.2%, male sex, Eastern Cooperative Oncology Group performance status ≥ 2, stage II versus I disease, and Rx/1 resection were prognostic. ASP remained prognostic for stage II disease (optimal,19.5%; PFS, 12 vs. 47 months). Log-rank test for ASP was significant at any cutoff ≥ 18% (OS) or from 9% to 59% (PFS).ASP was validated as prognostic factor for PFS and OS in patients with NSCLC and curative treatment intent, especially stage II. High ASP in stage II could imply intensified treatment or intensified follow-up.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (N = 496) and a validation set (DKTK MASTER cohort, N = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately re-analyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

2524 Background: The phase I GATTO study explored the feasibility, tolerability and preliminary activity of combining gatipotuzumab (GAT), a novel humanized monoclonal antibody binding to the tumor-associated epitope of mucin-1 (TA-MUC1) and an anti-EGFR antibody. Preclinical evidence suggests a complex interaction between TA-MUC1 and EGFR on the cell surface of epithelial tumors driving carcinogenesis processes and synergistic antibody dependent cell cytotoxicity activity with the dual targeting. Methods: Initially the study enrolled in a primary phase (PP) 20 patients with EGFR positive metastatic solid tumors, for whom no standard treatment was available. The first 6 patients were enrolled into a safety run-in phase and the number of dose-limiting toxicities (DLTs) was evaluated, in order to de-escalate the doses if needed. Patients received GAT administered at 1400 mg Q2W in combination with the glyco-optimized anti-EGFR antibody tomuzotuximab (TOM) at 1200 mg Q2W. Due to the risk of infusion related reactions (IRR), the first dose of TOM was reduced to 720 mg split over 2 consecutive days and three cycles of TOM monotherapy were given before start of treatment with GAT. As this regimen was proven safe, no DLT was observed and the initial dose remained unchanged, the study was amended to enroll in an expansion phase (EP) 30 additional patients with refractory colorectal cancer (CRC), non-small cell lung cancer (NSCLC), head and neck and breast cancers. TOM and GAT were given at the same doses and GAT treatment started already one week after the first dose of the anti-EGFR antibody. Additionally investigator had the choice to use a commercial anti-EGFR antibody in place of TOM. Results: By the time of the final analysis in January 2021, 52 refractory patients were enrolled and 50 received at least one dose of both GAT and anti-EGFR antibodies. Panitumumab (PAN) was used in 9 CRC patients. Because of the difference in treatment schedule, results are summarized separately for the 20 and 30 patients in PP and EP. Overall, the combined treatment was well tolerated and no DLT was observed in the whole study, nor related SAE or death. There were no treatment emergent adverse events (TEAEs) leading to dose interruptions or reductions in the PP and 2/30 (6.7%) patients in EP stopped both TOM and GAT. 16 IRRs were reported in 8/20 (40%) PP patients, and 40 IRRs in 10 (33.3%) EP patients. Only one event of chills was severe and only 6 events were related to GAT in the EP, all others to TOM. Other frequent TEAEs were those commonly observed with anti-EGFR treatment such as skin toxicity in 17 (85%) PP and 26 (86.7%) EP patients and hypomagnesemia in 10 (50%) PP and 7 (23.3%) EP patients. Conclusions: Combination of TA-MUC1 and EGFR targeting antibody is safe and feasible. Future studies should test this combination together with chemotherapy Clinical trial information: NCT03360734.