Publications by authors named "Sebastian Ochsenreither"

39 Publications

Carcinoma of Unknown Primary and the 8th Edition TNM Classification for Head and Neck Cancer.

Laryngoscope 2021 Mar 18. Epub 2021 Mar 18.

Department of Otorhinolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany.

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 < .001), UICC stage (TNM7, P < .001) and UICC stage (TNM8, P < .001) had a significant impact on survival in the univariate analysis. The 8th TNM classification resulted in a downstaging of p16-positive CUP syndromes and an upstaging of p16-negative syndromes.

Conclusion: The 8th TNM classification shows the lower UICC stage in p16-positive CUP syndromes. The prognostic significance for survival has improved from the 7th to the 8th TNM classification. LEVEL OF EVIDENCE USING THE 2011 OCEBM: Level 3. Laryngoscope, 2021.
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http://dx.doi.org/10.1002/lary.29499DOI Listing
March 2021

KRAS/TP53 co-mutations identify long-term responders to first line palliative treatment with pembrolizumab monotherapy in PD-L1 high (≥50%) lung adenocarcinoma.

Transl Lung Cancer Res 2021 Feb;10(2):737-752

Klinik für Pneumologie - Evangelische Lungenklinik Berlin Buch, Berlin, Germany.

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% 65%, P=0.13). Objective response rate (ORR), median progression-free survival (PFS) and overall survival (OS) in the KRAS group (n=32, 51.6%) were 63.3%, 19.8 months (mo.) and not estimable (NE), respectively. Results in KRAS and wild type patients were similar and by far lower (42.7%, P=0.06; 6.2 mo., P<0.001; 23.4 mo., P=0.08). TP53 mutations alone had no impact on response and survival. However, KRAS/TP53 co-mutations (n=12) defined a subset of long-term responders (ORR 100.0%, PFS 33.3 mo., OS NE). In contrast, patients with KRAS/TP53 mutations showed a dismal prognosis (ORR 27.3%, P=0.002; PFS 3.9 mo., P=0.001, OS 9.7 mo., P=0.02).

Conclusions: A comprehensive assessment of KRAS subtypes and TP53 mutations allows a highly relevant prognostic differentiation of patients with metastatic, PD-L1 high LuAD treated upfront with pembrolizumab.
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http://dx.doi.org/10.21037/tlcr-20-958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947421PMC
February 2021

Disulfiram Acts as a Potent Radio-Chemo Sensitizer in Head and Neck Squamous Cell Carcinoma Cell Lines and Transplanted Xenografts.

Cells 2021 Feb 28;10(3). Epub 2021 Feb 28.

Department of Otolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin Institute of Health, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany.

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/Cu inhibited the cell proliferation (inhibitory concentration 50 (IC) of DSF and DSF/Cu were 13.96 μM and 0.24 μM). DSF and cisplatin displayed a synergistic effect (CI values were < 1). DSF or DSF/Cu abolished the cisplatin-induced G2/M arrest (from 52.9% to 40.7% and 41.1%), and combining irradiation (IR) with DSF or DSF/Cu reduced the colony formation and attenuated the G2/M arrest (from 53.6% to 40.2% and 41.9%). The combination of cisplatin, DSF or DSF/Cu, and IR enhanced the radio-chemo sensitivity by inducing apoptosis (42.04% and 32.21%) and ROS activity (46.3% and 37.4%). DSF and DSF/Cu enhanced the sensitivity of HNSCC to cisplatin and IR. Confirming the initial data from patient-derived tumor xenograft (PDX) supported a strong rationale to repurpose DSF as a radio-chemosensitizer and to assess its therapeutic potential in a clinical setting.
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http://dx.doi.org/10.3390/cells10030517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999545PMC
February 2021

Pembrolizumab as First-Line Palliative Therapy in PD-L1 Overexpressing (≥ 50%) NSCLC: Real-world Results with Special Focus on PS ≥ 2, Brain Metastases, and Steroids.

Clin Lung Cancer 2021 Feb 6. Epub 2021 Feb 6.

Klinik für Pneumologie-Evangelische Lungenklinik Berlin Buch, Berlin, Germany.

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.
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http://dx.doi.org/10.1016/j.cllc.2021.02.001DOI Listing
February 2021

Impact of Smoking on the Survival of Patients With High-risk HPV-positive HNSCC: A Meta-analysis.

In Vivo 2021 Mar-Apr;35(2):1017-1026

Department of Otorhinolaryngology, Head and Neck Surgery (CVK and CCM), Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany;

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.
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http://dx.doi.org/10.21873/invivo.12345DOI Listing
October 2020

Pemetrexed-Based Chemotherapy Is Inferior to Pemetrexed-Free Regimens in Thyroid Transcription Factor 1 (TTF-1)-Negative, EGFR/ALK-Negative Lung Adenocarcinoma: A Propensity Score Matched Pairs Analysis.

Clin Lung Cancer 2020 11 22;21(6):e607-e621. Epub 2020 May 22.

Department of Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany; Division of Pulmonary Inflammation, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Introduction: 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.

Patients And Methods: 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.

Results: 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).

Conclusion: TTF-1 negativity is associated with a distinct cancer phenotype. Incorporation of this biomarker may be helpful when choosing an appropriate therapy regimen.
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http://dx.doi.org/10.1016/j.cllc.2020.05.014DOI Listing
November 2020

Distinct immune evasion in APOBEC-enriched, HPV-negative HNSCC.

Int J Cancer 2020 10 18;147(8):2293-2302. Epub 2020 Jun 18.

Department of Hematology and Oncology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Hindenburgdamm 30, Berlin, 12203, Germany.

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.
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http://dx.doi.org/10.1002/ijc.33123DOI Listing
October 2020

Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells.

Mol Carcinog 2020 07 25;59(7):724-735. Epub 2020 Apr 25.

Department of Pediatric Oncology and Hematology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt - Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

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.
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http://dx.doi.org/10.1002/mc.23202DOI Listing
July 2020

Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo.

Cancer Immunol Immunother 2020 Jul 10;69(7):1217-1227. Epub 2020 Mar 10.

Department of Hematology and Oncology, Campus Benjamin Franklin, Charité Berlin, Berlin, Germany.

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.
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http://dx.doi.org/10.1007/s00262-020-02519-6DOI Listing
July 2020

Support of a molecular tumour board by an evidence-based decision management system for precision oncology.

Eur J Cancer 2020 03 23;127:41-51. Epub 2020 Jan 23.

Charité Comprehensive Cancer Center, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Charitéplatz 1, 10117, Berlin, Germany; Department of Hematology and Oncology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Hindenburgdamm 30, 12203 Berlin, Germany; Berlin Institute of Health (BIH), Anna-Louisa-Karsch-Straße 2, 10178, Berlin, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejca.2019.12.017DOI Listing
March 2020

Validation of Independent Prognostic Value of Asphericity of F-Fluorodeoxyglucose Uptake in Non-Small-Cell Lung Cancer Patients Undergoing Treatment With Curative Intent.

Clin Lung Cancer 2020 05 15;21(3):264-272.e6. Epub 2019 Oct 15.

Department of Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Background: 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.

Patients And Methods: A retrospective study was performed of 311 NSCLC patients undergoing F-fluorodeoxyglucose positron emission tomography / computed tomography (F-FDG PET/CT) before curatively intended treatment (always including surgery). A total of 140 patients had International Union Against Cancer (UICC) stage I disease, 78 had stage II disease, and 93 had stage III disease (adenocarcinoma, n = 153; squamous-cell carcinoma, n = 141). Primary tumor MTV was delineated with semiautomated background-adapted threshold relative to the standardized maximum uptake value (SUV). Cox regression (progression-free survival [PFS] and overall survival [OS]) analysis for positron emission tomography (MTV, ASP, SUV) as well as for clinical (T/N descriptor, UICC stages), histologic, and treatment variables (Rx/1 vs. R0 resection, chemotherapy/radiotherapy yes/no) were performed.

Results: Events (progression and relapse) occurred in 167 of 311 patients; 137 died (median survivor follow-up, 37 months). In multivariable Cox regression for OS, ASP > 33.3% (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, ASP > 21.2%, 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).

Conclusion: 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.
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http://dx.doi.org/10.1016/j.cllc.2019.10.001DOI Listing
May 2020

Treatment of refractory germ-cell tumours with single-agent cabazitaxel: a German Testicular Cancer Study Group case series.

J Cancer Res Clin Oncol 2020 Feb 14;146(2):449-455. Epub 2019 Dec 14.

Haematology and Bone Marrow Transplantation with Division of Pneumology, Department of Oncology, University Medical Centre Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

Purpose: 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.

Methods: 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.

Results: 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%).

Conclusion: 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.
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http://dx.doi.org/10.1007/s00432-019-03071-2DOI Listing
February 2020

Local ablative treatment for synchronous single organ oligometastatic lung cancer-A propensity score analysis of 180 patients.

Lung Cancer 2018 11 25;125:164-173. Epub 2018 Sep 25.

Klinik für Pneumologie - Evangelische Lungenklinik Berlin Buch, Berlin, Germany.

Introduction: Local ablative treatment (LAT) improves outcome in lung cancer with oligometastatic disease (OMD) and potentially leads to long term survival. The aim of this retrospective study was to evaluate and quantify the additional benefit of LAT in synchronous OMD and to further identify prognostic factors for survival.

Patients And Methods: A propensity score-matched pairs analysis was performed on a set of patient and disease variables in 180 patients, treated for synchronous single organ OMD including non small-cell and neuroendocrine lung cancer with ≤4 metastases between 2000 and 2016 in 3 lung cancer centers in Berlin, Germany. Patients either received LAT for all sites of disease (intervention group) by means of surgery or stereotactic radiotherapy, or standard chemotherapy, if necessary combined with a local treatment with palliative intent (control group).

Results: Median follow-up time was 32.2 and 18.8 months for the intervention and control group, respectively. Substantial benefits in median progression-free survival (PFS, 25.1 vs. 8.2 months; HR, 0.30; 95% CI, 0.21-0.43; p < 0.001) and overall survival (OS, 60.4 vs. 22.5 months; HR, 0.42; 95% CI, 0.28-0.62; p < 0.001) were associated with LAT. Histology of adenocarcinoma and T1a primaries also predicted a favorable prognosis concerning PFS and OS. More favorable nodal stage (N0-2 vs. 3) and solitary metastases were associated with an extended PFS, whereas initial ECOG-PS (0-1 vs. 2) predicted OS.

Conclusions: LAT was the strongest predictor for PFS and OS in OMD with ≤4 metastases. Survival in the control group identifies OMD as a subset of lung cancer with a generally more favorable prognosis.
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http://dx.doi.org/10.1016/j.lungcan.2018.09.021DOI Listing
November 2018

pN status predicts outcomes in surgically treated pT1-pT2 patients of various disease stages with squamous cell carcinoma of the head and neck: a 17-year retrospective single center cohort study.

Eur Arch Otorhinolaryngol 2018 Nov 29;275(11):2787-2795. Epub 2018 Aug 29.

Department of Otorhinolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Campus Benjamin Franklin, 12200, Berlin, Germany.

Objectives: The optimal treatment for a substantial proportion of patients with pT1-pT2 squamous cell carcinomas of the head and neck (SCCHN) remains to be refined. The extent of surgery, role and potential benefit of adjuvant treatment are to be balanced against therapy-induced side effects. We compared the outcomes of surgery with or without adjuvant radiotherapy (RT) or chemotherapy (CRT) and investigated the prognostic value of established clinicopathological parameters.

Methods: Data were retrospectively collected for 227 patients who were treated by surgery alone (n = 31), RT (n = 87) and CRT (n = 109) in a single center.

Results: Patients with stage I/II disease who had received adjuvant RT showed a better disease-free survival (DFS) (P = 0.04) than those who had received adjuvant CRT treatment. Conversely, patients with stage III/IV disease who had received CRT showed a better overall survival (OS) (P = 0.003) and DFS (P = 0.03) than those who had received surgery alone or adjuvant RT without chemotherapy. Survival analysis demonstrated that patients with pN0 to pN1 had better OS (P = 0.02), disease-specific survival (DSS) (P = 0.003), DFS (P = 0.02) and metastases free survival (MFS) (P = 0.002) compared to patients with pN2 to pN3. Multivariate analysis showed that the pN status was an independent factor for OS (P = 0.03), DSS (P = 0.04), relapse-free survival (P = 0.03), DFS (P = 0.03).

Conclusion: The pN status is the most important prognostic factor for pT1 to pT2 SCCHN. Adjuvant CRT was associated with significantly better survival outcomes in patients with pN1 and pN2-3 or more advanced stage, while adjuvant RT showed significantly better outcomes in patients with pN0.
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http://dx.doi.org/10.1007/s00405-018-5108-zDOI Listing
November 2018

Aldehyde dehydrogenase 1 isoenzyme expression as a marker of cancer stem cells correlates to histopathological features in head and neck cancer: A meta-analysis.

PLoS One 2017 7;12(11):e0187615. Epub 2017 Nov 7.

Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical University, Wenzhou, P.R. China.

There is a lack of predictive biomarkers that can identify patients with head and neck squamous cell carcinoma (HNSCC) who will experience treatment failure and develop drug resistance, recurrence, and metastases. Cancer stem-like cells (CSC) were identified as a subset of cells within the tumor in a variety of solid tumors including HNSCC. CSC are considered the tumor-initiating population responsible for recurrence or metastasis and are associated with therapy resistance. This meta-analysis including fourteen studies with altogether 1258 patients updates and summarizes all relevant data on the impact of ALDH1+ CSC on the prognosis of HNSCC and its association with clinicopathological parameters. ALDH1 expression is highly correlated with tumor differentiation (G3 vs. G1+G2; odds ratio = 2.85. 95% CI: 1.72-4.73, P<0.0001) and decreased overall survival (relative risk = 1.77. 95% CI: 1.41-2.22, P<0.0001) if one out of seven studies was excluded because of heterogeneity. These findings provide insights into the understanding of more aggressive tumor phenotypes and also suggest that the prognostic value provided by HNSCC-subtyping by CSC frequency warrant further clinical investigation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675382PMC
November 2017

Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

Nat Biotechnol 2017 Dec 6;35(12):1188-1195. Epub 2017 Nov 6.

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

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.
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http://dx.doi.org/10.1038/nbt.4004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722674PMC
December 2017

[Immunotherapy Against Head and Neck Cancer Stem Cells].

Laryngorhinootologie 2017 Apr 10;96(4):216-224. Epub 2017 May 10.

Klinik für Hals-, Nasen-, Ohrenheilkunde, Kopf- und Halschirurgie, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Berlin.

Immunotherapy against head and neck cancer stem cells Immunologic therapies like antibodies in solid tumors like squamous cell cancer of the head and neck are administered either alone or in combination with radiation and chemotherapy. Despite some respectable successes, the effect of this therapy reaches its limits due the ability of the tumor to escape the immune system. Cancer stem cells seem to play an important role in this process due to their intrinsic resistance to conventional therapy and the ability to regenerate tumor heterogeneity. This way they substantially contribute to the formation of recurrences and metastases. Therefore, future immunotherapies should target specifically this subpopulation, possibly in combination with other therapeutic modalities. In this review the immunologic features of cancer stem cells and their potential as target for immunotherapies is summarized.
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http://dx.doi.org/10.1055/s-0043-101698DOI Listing
April 2017

Buparlisib and paclitaxel in patients with platinum-pretreated recurrent or metastatic squamous cell carcinoma of the head and neck (BERIL-1): a randomised, double-blind, placebo-controlled phase 2 trial.

Lancet Oncol 2017 03 26;18(3):323-335. Epub 2017 Jan 26.

Head and Neck Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; University of Milan, Milan, Italy. Electronic address:

Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation in squamous cell carcinoma of the head and neck contributes to treatment resistance and disease progression. Buparlisib, a pan-PI3K inhibitor, has shown preclinical antitumour activity and objective responses in patients with epithelial malignancies. We assessed whether the addition of buparlisib to paclitaxel improves clinical outcomes compared with paclitaxel and placebo in patients with recurrent or metastatic squamous cell carcinoma of the head and neck.

Methods: In this multicentre, randomised, double-blind, placebo-controlled phase 2 study (BERIL-1), we recruited patients aged 18 years and older with histologically or cytologically confirmed recurrent and metastatic squamous cell carcinoma of the head and neck after disease progression on or after one previous platinum-based chemotherapy regimen in the metastatic setting. Eligible patients were enrolled from 58 centres across 18 countries and randomly assigned (1:1) to receive second-line oral buparlisib (100 mg once daily) or placebo, plus intravenous paclitaxel (80 mg/m on days 1, 8, 15, and 22) in 28 day treatment cycles. Randomisation was done via a central patient screening and randomisation system with an interactive (voice and web) response system and stratification by number of previous lines of therapy in the recurrent and metastatic setting and study site. Patients and investigators (including local radiologists) were masked to treatment assignment from randomisation until the final overall survival analysis. The primary endpoint was progression-free survival by local investigator assessment per Response Evaluation Criteria In Solid Tumors (version 1.1) in all randomly assigned patients. Efficacy analyses were done on the intention-to-treat population, whereas safety was analysed in all patients who received at least one dose of study drug and had at least one post-baseline safety assessment according to the treatment they received. This trial is registered with ClinicalTrials.gov, number NCT01852292, and is ongoing but no longer enrolling patients.

Findings: Between Nov 5, 2013, and May 5, 2015, 158 patients were enrolled and randomly assigned to receive either buparlisib plus paclitaxel (n=79) or placebo plus paclitaxel (n=79). Median progression-free survival was 4·6 months (95% CI 3·5-5·3) in the buparlisib group and 3·5 months (2·2-3·7) in the placebo group (hazard ratio 0·65 [95% CI 0·45-0·95], nominal one-sided p=0·011). Grade 3-4 adverse events were reported in 62 (82%) of 76 patients in the buparlisib group and 56 (72%) of 78 patients in the placebo group. The most common grade 3-4 adverse events (occurring in ≥10% of patients in the buparlisib group vs the placebo group) were hyperglycaemia (17 [22%] of 76 vs two [3%] of 78), anaemia (14 [18%] vs nine [12%]), neutropenia (13 [17%] vs four [5%]), and fatigue (six [8%] vs eight [10%]). Serious adverse events (regardless of relation to study treatment) were reported for 43 (57%) of 76 patients in the buparlisib group and 37 (47%) of 78 in the placebo group. On-treatment deaths occurred in 15 (20%) of 76 patients in the buparlisib group and 17 (22%) of 78 patients in the placebo group; most were caused by disease progression and none were judged to be related to study treatment.

Interpretation: On the basis of the improved clinical efficacy with a manageable safety profile, the results of this randomised phase 2 study suggest that buparlisib in combination with paclitaxel could be an effective second-line treatment for patients with platinum-pretreated recurrent or metastatic squamous cell carcinoma of the head and neck. Further phase 3 studies are warranted to confirm this phase 2 finding.

Funding: Novartis Pharmaceuticals Corporation.
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http://dx.doi.org/10.1016/S1470-2045(17)30064-5DOI Listing
March 2017

Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix.

Oncotarget 2016 Nov;7(46):75379-75393

Charité Comprehensive Cancer Center, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression.A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma.A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC.Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition.
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http://dx.doi.org/10.18632/oncotarget.12211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342748PMC
November 2016

Cancer-testis antigen cyclin A1 is broadly expressed in ovarian cancer and is associated with prolonged time to tumor progression after platinum-based therapy.

BMC Cancer 2015 Oct 24;15:784. Epub 2015 Oct 24.

Department of Hematology, Oncology and Tumor Immunology - University Hospital Berlin, 12200, Berlin, Germany.

Background: Cyclin A1 is essential for male gametopoiesis. In acute myeloid leukemia, it acts as a leukemia-associated antigen. Cyclin A1 expression has been reported in several epithelial malignancies, including testicular, endometrial, and epithelial ovarian cancer (EOC). We analyzed Cyclin A1 expression in EOC and its correlation with clinical features to evaluate Cyclin A1 as a T-cell target in EOC.

Methods: Cyclin A1 mRNA expression in EOC and healthy tissues was quantified by microarray analysis and quantitative real-time PCR (qRT-PCR). Protein expression in clinical samples was assessed by immunohistochemistry (IHC) and was correlated to clinical features.

Results: Cyclin A1 protein was homogeneously expressed in 43 of 62 grade 3 tumor samples and in 1 of 10 grade 2 specimens (p < 0.001). Survival analysis showed longer time to progression (TTP) among patients with at least moderate Cyclin A1 expression (univariate: p = 0.018, multivariate: p = 0.035). FIGO stage, grading, age, macroscopic residual tumor after debulking, and peritoneal carcinomatosis / distant metastasis had no impact on TTP or overall survival (OS).

Conclusion: Cyclin A1 is highly expressed in most EOCs. The mechanism behind the prolonged TTP in patients with high Cyclin A1 expression warrants further investigation. The frequent, selectively high expression of Cyclin A1 in EOC makes it a promising target for T-cell therapies.
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http://dx.doi.org/10.1186/s12885-015-1824-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619521PMC
October 2015

Prophylaxis and management of venous thromboembolism in patients with myeloproliferative neoplasms: consensus statement of the Haemostasis Working Party of the German Society of Hematology and Oncology (DGHO), the Austrian Society of Hematology and Oncology (ÖGHO) and Society of Thrombosis and Haemostasis Research (GTH e.V.).

Ann Hematol 2014 Dec 14;93(12):1953-63. Epub 2014 Oct 14.

Department of Hematology and Oncology, Charite Berlin, Berlin, Hindenburgdamm 30, 12200, Berlin, Germany,

Patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) like polycythemia vera and essential thrombocythemia are at increased risk of arterial and venous thrombosis. Strategies of prevention may consist of platelet aggregation inhibitors and/or cytoreductive agents depending on the underlying disease and the individual risk. Clinical evidence for management of acute venous thromboembolic events in MPN patients is limited. Modality and duration of therapeutic anticoagulation after venous thrombosis has to be evaluated critically with special regard to the increased risk for spontaneous bleeding events associated with the underlying diseases. Both for therapy of the acute event and for secondary prophylaxis, low-molecular-weight heparins should preferentially be used. A prolongation of the therapeutic anticoagulation beyond the usual 3 to 6 months can only be recommended in high-risk settings and after careful evaluation of potential risks and benefits for the individual patient. New direct oral anticoagulants (NOAC) should not preferentially be used due to lack of clinical experience in patients with MPN and potential drug interactions (e.g. with JAK inhibitors). Consequent treatment of the underlying myeloproliferative disease and periodical evaluation of the response to therapy is crucial for optimal secondary prophylaxis of thromboembolic events in those patients.
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http://dx.doi.org/10.1007/s00277-014-2224-8DOI Listing
December 2014

Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo.

Mol Ther Nucleic Acids 2013 Nov 19;2:e134. Epub 2013 Nov 19.

Laboratory of Molecular & Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium.

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.
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http://dx.doi.org/10.1038/mtna.2013.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889186PMC
November 2013

Characterization of small spheres derived from various solid tumor cell lines: are they suitable targets for T cells?

Clin Exp Metastasis 2013 Aug 22;30(6):781-91. Epub 2013 Mar 22.

Department of Medicine III, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin, Germany,

T cell based immunotherapy has been investigated in a variety of malignancies and analyses have been mostly founded on in vitro data with tumor cell monolayers. However, three-dimensional (3D) culture models might mimic more closely the 'in vivo' conditions than 2D monolayers. Therefore, we analyzed the expression of tumor-associated antigens (TAA) and of molecules involved in antigen processing and presentation (APM) in tumor spheres, which served as an in vitro model for micrometastasis which might be enriched in tumor propagating cancer stem cells. For enrichment of sphere cells 12 human solid tumor cell lines were cultured in serum-free medium. Expression of a variety of TAA and APM were analyzed by RT-PCR and/or flow cytometry and compared to expression in corresponding adherent bulk cells grown in regular growth medium. Compared to adherent cells, spheres showed equal or higher mRNA expression levels of LMP2, LMP7 and MECL-1, of TAP1 and TAP2 transporters and, surprisingly, also of TAA including differentiation antigens. However, downregulation or loss of HLA-I and HLA-II molecules in spheres was observed in 8 of 10 and 1 of 2 cell lines, respectively, and was unresponsive to stimulation with IFN-γ. Although tumor spheres express TAA and molecules of intracellular antigen processing, they are defective in antigen presentation due to downregulation of HLA surface expression which may lead to immune evasion.
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http://dx.doi.org/10.1007/s10585-013-9578-5DOI Listing
August 2013

Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients.

Sci Transl Med 2013 Feb;5(174):174ra27

Program in Immunology, Fred Hutchinson Cancer Research Center (FHCRC), Seattle, WA 98109, USA.

Relapse remains a leading cause of death after allogeneic hematopoietic cell transplantation (HCT) for patients with high-risk leukemias. The potentially beneficial donor T cell-mediated graft-versus-leukemia (GVL) effect is often mitigated by concurrent graft-versus-host disease (GVHD). Providing T cells that can selectively target Wilms tumor antigen 1 (WT1), a transcription factor overexpressed in leukemias that contributes to the malignant phenotype, represents an opportunity to promote antileukemic activity without inducing GVHD. HLA-A*0201-restricted WT1-specific donor-derived CD8 cytotoxic T cell (CTL) clones were administered after HCT to 11 relapsed or high-risk leukemia patients without evidence of on-target toxicity. The last four treated patients received CTL clones generated with exposure to interleukin-21 (IL-21) to prolong in vivo CTL survival, because IL-21 can limit terminal differentiation of antigen-specific T cells generated in vitro. Transferred cells exhibited direct evidence of antileukemic activity in two patients: a transient response in one patient with advanced progressive disease and the induction of a prolonged remission in a patient with minimal residual disease (MRD). Additionally, three treated patients at high risk for relapse after HCT survive without leukemia relapse, GVHD, or additional antileukemic treatment. CTLs generated in the presence of IL-21, which were transferred in these latter three patients and the patient with MRD, all remained detectable long-term and maintained or acquired in vivo phenotypic and functional characteristics associated with long-lived memory CD8 T cells. This study supports expanding efforts to immunologically target WT1 and provides insights into the requirements necessary to establish potent persistent T cell responses.
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http://dx.doi.org/10.1126/scitranslmed.3004916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678970PMC
February 2013

Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen.

Blood 2012 Jun 23;119(23):5492-501. Epub 2012 Apr 23.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1-specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell-based therapy approaches.
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http://dx.doi.org/10.1182/blood-2011-07-365890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3369684PMC
June 2012

Wilms' tumor protein 1 (WT1) peptide vaccination in AML patients: predominant TCR CDR3β sequence associated with remission in one patient is detectable in other vaccinated patients.

Cancer Immunol Immunother 2012 Mar 7;61(3):313-22. Epub 2011 Sep 7.

Department of Hematology and Oncology, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin, Germany.

Background: Clinically effective T-cell responses can be elicited by single peptide vaccination with Wilms' tumor 1 (WT1) epitope 126-134 in patients with acute myeloid leukemia (AML). We recently showed that a predominant T-cell receptor (TCR) β chain was associated with vaccine-induced complete remission in an AML patient (patient 1). In this study, we address the question of whether this predominant clone or the accompanying Vβ11 restriction could be found in other AML patients vaccinated with the same WT1 peptide.

Materials And Methods: For assessment of Vβ usage, cytotoxic T lymphocytes (CTLs) from four vaccinated patients were divided into specific and non-specific by epitope-specific enrichment. Vβ families were quantified in both fractions using reverse transcribed quantitative PCR. Vβ11-positive 'complementary determining region 3' (CDR3) sequences were amplified from these samples, from bone marrow samples of 17 other vaccination patients, and from peripheral blood of six healthy controls, cloned and sequenced.

Results: We observed a clear bias towards Vβ11 usage of the WT1-specific CTL populations in all four patients. The predominant CDR3β amino acid (AA) sequence of patient 1 was detected in two other patients. CDR3β loops with closely related AA sequences were only found in patient 1. There were no CDR3β AA sequences with side chains of identical chemical properties detected in any patient.

Conclusion: We provide the first data addressing TCR Vβ chain usage in WT1-specific T-cell populations after HLA A*0201-restricted single peptide vaccination. We demonstrate both shared Vβ restriction and the sharing of a TCR β transcript with proven clinical impact in one patient.
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http://dx.doi.org/10.1007/s00262-011-1099-yDOI Listing
March 2012

Immunomodulatory effects of sorafenib on peripheral immune effector cells in metastatic renal cell carcinoma.

Eur J Cancer 2011 Mar 5;47(5):690-6. Epub 2011 Jan 5.

Department of Medicine III (Hematology and Oncology), Charité, Campus Benjamin Franklin, Berlin, Germany.

Background: Tyrosine kinase inhibitors (TKI) such as sorafenib have substantially improved the prognosis of metastatic renal cell carcinoma (mRCC) patients, but long-term remissions have only been reached with immunotherapy. Sequencing or combining TKI treatment with immunotherapy may represent an attractive therapeutic concept. However, in vitro data have shown that TKI may not only affect tumour cells, but also inhibit signalling in immune effector cells. Therefore, we asked whether sorafenib had an influence on peripheral immune effector cells in a cohort of 35 mRCC patients receiving sorafenib treatment.

Methods: Peripheral blood (pB) samples were analysed at baseline and after 8 weeks of treatment. IL-10 and TGF-ß mRNA levels were quantified by RT-PCR; regulatory T cell (Treg) counts and intracellular cytokine responses (TNF-α, IFN-γ, IL-10 and TGF-ß) of mononuclear cell subsets were determined by flow cytometry after in vitro stimulation with PMA/ionomycin.

Results: Sorafenib did not alter the elevated TGF-ß and IL-10 mRNA levels or elevated frequencies of IL-10 and TGF-ß producing monocytes and had no influence on type 1 cytokine responses in pB. CD4+CD25(high) FOXP3+/CD3+ T cells, likely representing Treg cells, decreased during sorafenib therapy.

Conclusions: In vivo, sorafenib treatment was associated with a decrease in frequency of Treg cells without influencing the function of peripheral immune effector cells. Therefore, although sorafenib did not convert the immunosuppressive phenotype associated with mRCC, it seemed to be a possible candidate for combination with immunotherapy.
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http://dx.doi.org/10.1016/j.ejca.2010.11.021DOI Listing
March 2011

Systemic immune tuning in renal cell carcinoma: favorable prognostic impact of TGF-β1 mRNA expression in peripheral blood mononuclear cells.

J Immunother 2011 Jan;34(1):113-9

Department of Medicine III (Hematology and Oncology), Charité, Campus Benjamin Franklin, Berlin, Germany.

Renal cell carcinoma (RCC) can inhibit protective immunity by induction of immunosuppressive cells that produce inhibitory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. If this immunosuppression influences response to kinase inhibitors such as sorafenib is not known. Therefore, we asked for the prognostic influence of cells with immunosuppressive properties in peripheral blood (pB) in a cohort of metastatic clear cell renal cell carcinoma (mRCC) patients uniformly receiving sorafenib treatment. IL-10 and TGF-β mRNA levels, regulatory T-cell (Treg) counts, and frequencies of IL-10/TGF-β producing mononuclear cell subsets were determined in pB from 46 patients with mRCC before receiving sorafenib treatment. Relationship between established clinical and laboratory prognostic factors and outcome were examined by univariate and multivariate Cox regression analysis. IL-10 and TGF-β1 mRNA levels, and frequencies of CD4(+)CD25high/CD3(+) and CD4(+)CD25highFoxP3(+)/CD3(+)Treg cells were significantly higher in mRCC patients compared with healthy individuals. Monocytes were suggested as main producers of IL-10 and TGF-β. In a multivariate analysis low ECOG score and-surprisingly-high TGF-β1 mRNA levels were independently associated with favorable progression-free survival (P=0.005 and P=0.003, respectively) and overall survival (P=0.001 and P=0.039, respectively). In conclusion, mRCC is associated with an immunosuppressive phenotype in peripheral blood. The positive prognostic influence of high TGF-β1 mRNA expression levels may reflect immune promoting functions of TGF-β in combined activity with inflammatory cytokines.
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http://dx.doi.org/10.1097/CJI.0b013e3181fb6580DOI Listing
January 2011

"Wilms Tumor Protein 1" (WT1) peptide vaccination-induced complete remission in a patient with acute myeloid leukemia is accompanied by the emergence of a predominant T-cell clone both in blood and bone marrow.

J Immunother 2011 Jan;34(1):85-91

Department of Hematology, Oncology, Charité Campus Benjamin Franklin, Berlin, Germany.

Within the last few years, the first peptide vaccination trials for treatment of acute myeloid leukemia (AML) have been initiated. Athough the presence of epitope-specific T cells could be seen both in bone marrow (BM) and peripheral blood (PB), nothing is known about their clonal composition. In this study, we analyzed material from a patient with recurrent AML vaccinated with "Wilms Tumor Protein 1" (WT1) peptide, who achieved a complete remission (CR) lasting for 12 months. For identification of expanded WT1-specific T-cell clones, enrichment by tetramer and IFNγ secretion were followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR Vβ-families. Vβ-families with increase in the enriched fraction were cloned and sequenced. A predominant clone was quantified by clonotypic qRT PCR from PB and BM. Quantity and functionality of WT1-specific cells were assessed by tetramer analyses and intracellular IFNγ staining. A specific predominant clone was identified during clinical remission. Clone-specific qRT PCR showed an increase both in PB and BM after 8 vaccinations. Six months after achieving CR, the transcript levels in BM decreased. Relapse was accompanied by secondary rise of the WT1-specific clone in PB but not in BM. In parallel, a lack of vaccine-induced WT1 specific IFNγ production was observed at that timepoint. In conclusion, we provide first data regarding evolution and compartmentalization of a peptide vaccine-induced T-cell clone in PB and BM of an AML patient. At the time of relapse, the same clone reappeared spontaneously in PB but not in BM showing impaired functionality.
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http://dx.doi.org/10.1097/CJI.0b013e3181f3cc5cDOI Listing
January 2011

Expression of the stem cell markers nestin and CD133 on circulating melanoma cells.

J Invest Dermatol 2011 Feb 30;131(2):487-94. Epub 2010 Sep 30.

Department of Hematology and Medical Oncology, Charité, Campus Benjamin Franklin, Berlin, Germany.

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells, presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study, 20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02), number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02), tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01), and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis.
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http://dx.doi.org/10.1038/jid.2010.285DOI Listing
February 2011