Publications by authors named "Ruth Seggewiss-Bernhardt"

19 Publications

  • Page 1 of 1

Amplification in Non-Small Cell Lung Cancer (NSCLC)-A Consecutive Evaluation Using Next-Generation Sequencing (NGS) in a Real-World Setting.

Cancers (Basel) 2021 Oct 7;13(19). Epub 2021 Oct 7.

Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany.

In non-small cell lung cancer (NSCLC), approximately 1-3% of cases harbor an increased gene copy number (GCN) of the gene. This alteration can be due to de novo amplification of the gene or can represent a secondary resistance mechanism in response to targeted therapies. To date, the gold standard method to evaluate the GCN of is fluorescence in situ hybridization (FISH). However, next-generation sequencing (NGS) is becoming more relevant to optimize therapy by revealing the mutational profile of each NSCLC. Using evaluable = 205 NSCLC cases of a consecutive cohort, this study addressed the question of whether an amplicon based NGS assay can completely replace the FISH method regarding the classification of GCN status. Out of the 205 evaluable cases, only = 9 cases (43.7%) of = 16 high-level amplified cases assessed by FISH were classified as amplified by NGS. Cases harboring a GCN > 10 showed the best concordance when comparing FISH versus NGS (80%). This study confirms that an amplicon-based NGS assessment of the GCN detects high-level amplified cases harboring a GCN > 10 but fails to detect the various facets of gene amplification in the context of a therapy-induced resistance mechanism.
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http://dx.doi.org/10.3390/cancers13195023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508248PMC
October 2021

Preinfection laboratory parameters may predict COVID-19 severity in tumor patients.

Cancer Med 2021 07 13;10(13):4424-4436. Epub 2021 Jun 13.

Klinik für Hämatologie, Medizinische Onkologie und Palliativmedizin, Marien Kliniken Siegen, Siegen, Germany.

Background: Infection with SARS-CoV-2 leads to COVID-19, the course of which is highly variable and depends on numerous patient-specific risk factors. Patients with tumor diseases are considered to be more susceptible to severe COVID-19; however, they also represent a heterogeneous group of individuals with variable risk. Identifying specific risk factors for a severe course of COVID-19 in patients with cancer is of great importance.

Methods: Patients diagnosed with solid tumors or hematological malignancies and PCR-confirmed SARS-CoV-2 infection were included into the multicentric ADHOK (Arbeitsgemeinschaft der Hämatologen und Onkologen im Krankenhaus e.V.) coronavirus tumor registry. Detailed information about the patients' cancer disease, treatment, and laboratory parameters prior to infection, was collected retrospectively. The outcome of the SARS-CoV-2 infection was graded according to the WHO.

Results: A total of 195 patients (68% with solid neoplasms and 32% with hematological malignancies) were included in the registry. Overall, the course of the SARS-CoV-2 infection varied greatly, as 69% of all patients were either asymptomatic or encountered a mild to moderate course, while 23% of the cohort died from COVID-19. In multivariable analysis, preinfection laboratory parameters (determined at least 10 days and a median of 21 days before the first documentation of SARS-CoV-2 infection) significantly correlated with severe course of the disease. Out of these, the absolute neutrophil count prior to infection showed the strongest association with COVID-19-related death.

Conclusion: The course of COVID-19 in patients with tumor diseases is highly variable. Preinfection laboratory parameters may aid to identify patients at risk for severe COVID-19 at an early stage prior to infection with the virus. German Clinical Trials Register identification: DRKS00023012.
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http://dx.doi.org/10.1002/cam4.4023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8267142PMC
July 2021

[Robot-assisted laparoscopic bladder diverticulum resection (RABDR)].

Aktuelle Urol 2021 May 11. Epub 2021 May 11.

Sozialstiftung Bamberg, Klinik für Urologie, Kinderurologie und roboterassistierte minimalinvasive Urologie, Bamberg.

Aims:  Since October 2018, urinary bladder diverticulum resections at our Department of Urology have been carried out with robot assistance and with minimal invasivion, Paediatrical urological and robot-assisted minimally invasive urological surgery for the Bamberg Social Foundation were performed with the DaVinci robotic system. The aim of the present study was to record the surgical results of our patients and to compare these if necessary with available data on optimal diagnostic and therapy.

Methods:  In this retrospective analysis, we included all patients who received RAHDR between October 2018 and March 2020. The primary endpoints were postoperative blood loss (postoperative haemoglobin decrease), the operation time (min), complications according to the Clavien-Dindo classification, length of hospital stay (days), postoperative residual urine, postoperative urine extravasation at the anastomosis of the bladder, postoperative quality of life and postoperative satisfaction with micturition.

Results:  We reviewed a total of 11 patients, all of whom were male. Mean age was 64.8 years (52-82). Average BMI was 26.5 (19-37). 3 patients were ranked with ASA score III, 5 with II and 3 with I. The average residual urine value preoperatively was 183 ml (90-240). A cystogram to rule out extravasation was performed on day 6 postoperatively. The mean duration of surgery was 212 min (148-294) and the mean duration of hospitalisation was 7.6 days (6-10). The mean residual urinary value after surgery was 25 ml (10-60). The mean postoperative maximum of flow was measured at 27.7 ml/s (11-55). No contrast agent extravasation in the cystogram was detected in any of the patients. The complications according to Clavien were not measurable. The mean postoperative haemoglobin decrease was 1.61 g/dl (0-3. 2).

Conclusions:  In most cases, the removal of one or more bladder diverticula is possible using the minimally invasive robotic technique. Various surgeries such as YV-bladder neck plastic, prostate adenoma enucleation, bladder stone restoration, and inguinal herniotomy can be carried out simultaneously. A robot-assisted urinary bladder diverticulum resection is an effective and gentle procedure. However, it must be considered that it brings financial disadvantages due to the lack of adequate representation within the German DRG-system (Diagnosis Related Groups).
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http://dx.doi.org/10.1055/a-1327-5509DOI Listing
May 2021

Pasotuxizumab, a BiTE immune therapy for castration-resistant prostate cancer: Phase I, dose-escalation study findings.

Immunotherapy 2021 02 10;13(2):125-141. Epub 2020 Nov 10.

Translational Oncology, Comprehensive Cancer Center Mainfranken, University Hospital Würzburg, Josef-Schneider-Str. 6, 97080 Würzburg, Germany.

 We report results of a first-in-human study of pasotuxizumab, a PSMA bispecific T-cell engager (BiTE) immune therapy mediating T-cell killing of tumor cells in patients with advanced castration-resistant prostate cancer. We assessed once-daily subcutaneous (SC) pasotuxizumab. All SC patients developed antidrug antibodies; therefore, continuous intravenous (cIV) infusion was assessed.  A total of 47 patients received pasotuxizumab (SC: n = 31, 0.5-172 μg/d; cIV: n = 16, 5-80 μg/d). The SC maximum tolerated dose was 172.0 μg/d. A sponsor change stopped the cIV cohort early; maximum tolerated dose was not determined. PSA responders occurred (>50% PSA decline: SC, n = 9; cIV, n = 3), including two long-term responders. Data support pasotuxizumab safety in advanced castration-resistant prostate cancer and represent evidence of BiTE monotherapy efficacy in solid tumors. Clinical trial registration: NCT01723475 (ClinicalTrials.gov).
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http://dx.doi.org/10.2217/imt-2020-0256DOI Listing
February 2021

[A rare case of renal ectopic thyroid tissue].

Aktuelle Urol 2021 Feb 23;52(1):64-66. Epub 2020 Sep 23.

Sozialstiftung Bamberg, Klinik für Urologie, Kinderurologie und roboterassistierte minimalinvasive Urologie, Bamberg.

We present a rare case of ectopic thyroid tissue found during robotic nephrectomy of a kidney with a suspected malignant tumour. Such cases of ectopic thyroid tissue are extremely rare in the literature. If ectopic thyroid tissue occurs, it is usually found in the neck region or in the upper mediastinum. Clinical symptoms depend on size, localisation and hormonal function of the ectopic tissue. Surgical resection remains the treatment of choice; in individual cases, conservative treatment can be an option. This case report aims to emphasise that renal tumours of unknown origin might be paraneoplastic or ectopic tissue of other organs.
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http://dx.doi.org/10.1055/a-1182-1961DOI Listing
February 2021

Keratinocyte growth factor impairs human thymic recovery from lymphopenia.

JCI Insight 2019 05 7;5. Epub 2019 May 7.

Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom.

Background: The lymphocyte-depleting antibody alemtuzumab is a highly effective treatment of relapsing-remitting multiple sclerosis (RRMS); however 50% of patients develop novel autoimmunity post-treatment. Most at risk are individuals who reconstitute their T-cell pool by proliferating residual cells, rather than producing new T-cells in the thymus; raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in non-human primates.

Methods: Following a dose-tolerability sub-study, individuals with RRMS (duration ≤10 years; expanded disability status scale ≤5·0; with ≥2 relapses in the previous 2 years) were randomised to placebo or 180mcg/kg/day palifermin, given for 3 days immediately prior to and after each cycle of alemtuzumab, with repeat doses at M1 and M3. The interim primary endpoint was naïve CD4+ T-cell count at M6. Exploratory endpoints included: number of recent thymic-emigrants (RTEs) and signal-joint T-cell receptor excision circles (sjTRECs)/mL of blood. The trial primary endpoint was incidence of autoimmunity at M30.

Findings: At M6, individuals receiving palifermin had fewer naïve CD4+T-cells (2.229x107/L vs. 7.733x107/L; p=0.007), RTEs (16% vs. 34%) and sjTRECs/mL (1100 vs. 3396), leading to protocol-defined termination of recruitment. No difference was observed in the rate of autoimmunity between the two groupsConclusion: In contrast to animal studies, palifermin reduced thymopoiesis in our patients. These results offer a note of caution to those using palifermin to promote thymopoiesis in other settings, particularly in the oncology/haematology setting where alemtuzumab is often used as part of the conditioning regime.

Trial Registration: ClinicalTrials.gov NCT01712945Funding: MRC and Moulton Charitable Foundation.
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http://dx.doi.org/10.1172/jci.insight.125377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629095PMC
May 2019

Genomic Profiling Identifies Outcome-Relevant Mechanisms of Innate and Acquired Resistance to Third-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy in Lung Cancer.

JCO Precis Oncol 2019 27;3. Epub 2019 Mar 27.

University Hospital of Cologne, Cologne, Germany.

Purpose: Third-generation epidermal growth factor receptor () tyrosine kinase inhibitors (TKIs) are effective in acquired resistance (AR) to early-generation EGFR TKIs in EGFR-mutant lung cancer. However, efficacy is marked by interindividual heterogeneity. We present the molecular profiles of pretreatment and post-treatment samples from patients treated with third-generation EGFR TKIs and their impact on treatment outcomes.

Methods: Using the databases of two lung cancer networks and two lung cancer centers, we molecularly characterized 124 patients with p.T790M-positive AR to early-generation EGFR TKIs. In 56 patients, correlative analyses of third-generation EGFR TKI treatment outcomes and molecular characteristics were feasible. In addition, matched post-treatment biopsy samples were collected for 29 patients with progression to third-generation EGFR TKIs.

Results: Co-occurring genetic aberrations were found in 74.4% of p.T790-positive samples (n = 124). Mutations in were the most frequent aberrations detected (44.5%; n = 53) and had no significant impact on third-generation EGFR TKI treatment. Mesenchymal-epithelial transition factor () amplifications were found in 5% of samples (n = 6) and reduced efficacy of third-generation EGFR TKIs significantly (eg, median progression-free survival, 1.0 months; 95% CI, 0.37 to 1.72 8.2 months; 95% CI, 1.69 to 14.77 months; ≤ .001). Genetic changes in the 29 samples with AR to third-generation EGFR TKIs were found in (eg, p.T790M loss, acquisition of p.C797S or p.G724S) or in other genes (eg, amplification, mutations).

Conclusion: Additional genetic aberrations are frequent in EGFR-mutant lung cancer and may mediate innate and AR to third-generation EGFR TKIs. amplification was strongly associated with primary treatment failure and was a common mechanism of AR to third-generation EGFR TKIs. Thus, combining EGFR inhibitors with TKIs targeting common mechanisms of resistance may delay AR.
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http://dx.doi.org/10.1200/PO.18.00210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446436PMC
March 2019

Alpelisib Plus Fulvestrant in PIK3CA-Altered and PIK3CA-Wild-Type Estrogen Receptor-Positive Advanced Breast Cancer: A Phase 1b Clinical Trial.

JAMA Oncol 2019 02 14;5(2):e184475. Epub 2019 Feb 14.

Department of Medicine, University of California at San Francisco, Helen Diller Family Comprehensive Cancer Center, San Francisco.

Importance: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in patients with estrogen receptor-positive (ER+), endocrine therapy-resistant breast cancers.

Objective: To assess the maximum tolerated dose (MTD), safety, and activity of alpelisib, an oral, PI3Kα-specific inhibitor, plus fulvestrant in patients with ER+ advanced breast cancer (ABC).

Design, Setting, And Participants: An open-label, single-arm, phase 1b study of alpelisib plus fulvestrant was conducted at 10 centers in 5 countries. Participants were 87 postmenopausal women with PIK3CA-altered or PIK3CA-wild-type ER+ ABC, whose cancer progressed during or after antiestrogen therapy. The study began enrolling patients October 5, 2010, and the data cutoff was March 22, 2017.

Interventions: Escalating doses of alpelisib were administered once daily, starting at 300 mg, plus fixed-dose fulvestrant, 500 mg, in the dose-escalation phase; alpelisib at the recommended phase 2 dose plus fulvestrant in the dose-expansion phase.

Main Outcomes And Measures: The primary end point was determination of the MTD of once-daily alpelisib plus fulvestrant. Secondary end points included safety and preliminary activity.

Results: From October 5, 2010, to March 22, 2017, 87 women (median age: 58 years [range, 37-79 years]; median of 5 prior lines of antineoplastic therapy) received escalating once-daily doses of alpelisib (300 mg, n = 9; 350 mg, n = 8; 400 mg, n = 70) plus fixed-dose fulvestrant (500 mg). During dose escalation, dose-limiting toxic effects were reported in 1 patient (alpelisib, 400 mg): diarrhea (grade 2), vomiting, fatigue, and decreased appetite (all grade 3). The MTD of alpelisib when combined with fulvestrant was 400 mg once daily, and the recommended phase 2 dose was 300 mg once daily. Overall, the most frequent grade 3/4 adverse events with alpelisib, 400 mg, once daily (≥10% of patients), regardless of causality, were hyperglycemia (19 [22%]) and maculopapular rash (11 [13%]); 9 patients permanently discontinued therapy owing to adverse events. Median progression-free survival at the MTD was 5.4 months (95% CI, 4.6-9.0 months). Median progression-free survival with alpelisib, 300 to 400 mg, once daily plus fulvestrant was longer in patients with PIK3CA-altered tumors (9.1 months; 95% CI, 6.6-14.6 months) vs wild-type tumors (4.7 months; 95% CI, 1.9-5.6 months). Overall response rate in the PIK3CA-altered group was 29% (95% CI, 17%-43%), with no objective tumor responses in the wild-type group.

Conclusions And Relevance: Alpelisib plus fulvestrant has a manageable safety profile in patients with ER+ ABC, and data suggest that this combination may have greater clinical activity in PIK3CA-altered vs wild-type tumors.

Trial Registration: ClinicalTrials.gov identifier: NCT01219699.
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http://dx.doi.org/10.1001/jamaoncol.2018.4475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439561PMC
February 2019

A multicenter phase 1 study of solitomab (MT110, AMG 110), a bispecific EpCAM/CD3 T-cell engager (BiTE®) antibody construct, in patients with refractory solid tumors.

Oncoimmunology 2018;7(8):e1450710. Epub 2018 Apr 18.

Department of Oncology/Hematology, Bone Marrow Transplantation and Section Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

We assessed the tolerability and antitumor activity of solitomab, a bispecific T-cell engager (BiTE®) antibody construct targeting epithelial cell adhesion molecule (EpCAM). Patients with relapsed/refractory solid tumors not amenable to standard therapy received solitomab as continuous IV infusion in a phase 1 dose-escalation study with six different dosing schedules. The primary endpoint was frequency and severity of adverse events (AEs). Secondary endpoints included pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity. Sixty-five patients received solitomab at doses between 1 and 96 µg/day for ≥28 days. Fifteen patients had dose-limiting toxicities (DLTs): eight had transient abnormal liver parameters shortly after infusion start or dose escalation (grade 3, n = 4; grade 4, n = 4), and one had supraventricular tachycardia (grade 3); all events resolved with solitomab discontinuation. Six patients had a DLT of diarrhea: four events resolved (grade 3, n = 3; grade 4, n = 1), one (grade 3) was ongoing at the time of treatment-unrelated death, and one (grade 3) progressed to grade 5 after solitomab discontinuation. The maximum tolerated dose was 24 µg/day. Overall, 95% of patients had grade ≥3 treatment-related AEs, primarily diarrhea, elevated liver parameters, and elevated lipase. Solitomab half-life was 4.5 hours; serum levels plateaued within 24 hours. One unconfirmed partial response was observed. In this study of a BiTE® antibody construct targeting solid tumors, treatment of relapsed/refractory EpCAM-positive solid tumors with solitomab was associated with DLTs, including severe diarrhea and increased liver enzymes, which precluded dose escalation to potentially therapeutic levels.
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http://dx.doi.org/10.1080/2162402X.2018.1450710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136859PMC
April 2018

Phosphatidylinositol 3-Kinase α-Selective Inhibition With Alpelisib (BYL719) in PIK3CA-Altered Solid Tumors: Results From the First-in-Human Study.

J Clin Oncol 2018 05 5;36(13):1291-1299. Epub 2018 Feb 5.

Dejan Juric, Massachusetts General Hospital Cancer Center, Boston; Alan Huang, Novartis Institutes for BioMedical Research, Cambridge, MA; Jordi Rodon and Josep Tabernero, Vall d'Hebron University Hospital and Institute of Oncology, Universitat Autònoma de Barcelona; Marta Gil-Martin, Catalan Institute of Oncology - Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain; Filip Janku, The University of Texas MD Anderson Cancer Center, Houston, TX; Howard A. Burris, Sarah Cannon Research Institute and Tennessee Oncology; Jordan Berlin, Vanderbilt-Ingram Cancer Center, Nashville, TN; Jan H.M. Schellens, Netherlands Cancer Institute, Amsterdam, the Netherlands; Mark R. Middleton, National Institute for Health Research, Oxford Biomedical Research Centre, Churchill Hospital, Oxford, United Kingdom; Martin Schuler, West German Cancer Center, University Duisburg-Essen, and German Cancer Consortium, Partner Site University Hospital Essen, Essen; Ruth Seggewiss-Bernhardt, Comprehensive Cancer Center Mainfranken, University Hospital Würzburg, Würzburg, Germany; Hope S. Rugo, University of California San Francisco Helen Diller Family Comprehensive Cancer Center, San Francisco, CA; Douglas Bootle, David Demanse, Lars Blumenstein, and Cornelia Quadt, Novartis Pharma AG, Basel, Switzerland; Christina Coughlin, Novartis Pharmaceuticals Corporation, East Hanover, NJ; and José Baselga, Memorial Sloan Kettering Cancer Center, New York, NY.

Purpose We report the first-in-human phase Ia study to our knowledge ( ClinicalTrials.gov identifier: NCT01219699) identifying the maximum tolerated dose and assessing safety and preliminary efficacy of single-agent alpelisib (BYL719), an oral phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor. Patients and Methods In the dose-escalation phase, patients with PIK3CA-altered advanced solid tumors received once-daily or twice-daily oral alpelisib on a continuous schedule. In the dose-expansion phase, patients with PIK3CA-altered solid tumors and PIK3CA-wild-type, estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer received alpelisib 400 mg once daily. Results One hundred thirty-four patients received treatment. Alpelisib maximum tolerated doses were established as 400 mg once daily and 150 mg twice daily. Nine patients (13.2%) in the dose-escalation phase had dose-limiting toxicities of hyperglycemia (n = 6), nausea (n = 2), and both hyperglycemia and hypophosphatemia (n = 1). Frequent all-grade, treatment-related adverse events included hyperglycemia (51.5%), nausea (50.0%), decreased appetite (41.8%), diarrhea (40.3%), and vomiting (31.3%). Alpelisib was rapidly absorbed; half-life was 7.6 hours at 400 mg once daily with minimal accumulation. Objective tumor responses were observed at doses ≥ 270 mg once daily; overall response rate was 6.0% (n = 8; one patient with endometrial cancer had a complete response, and seven patients with cervical, breast, endometrial, colon, and rectal cancers had partial responses). Stable disease was achieved in 70 (52.2%) patients and was maintained > 24 weeks in 13 (9.7%) patients; disease control rate (complete and partial responses and stable disease) was 58.2%. In patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer, median progression-free survival was 5.5 months. Frequently mutated genes (≥ 10% tumors) included TP53 (51.3%), APC (23.7%), KRAS (22.4%), ARID1A (13.2%), and FBXW7 (10.5%). Conclusion Alpelisib demonstrated a tolerable safety profile and encouraging preliminary activity in patients with PIK3CA-altered solid tumors, supporting the rationale for selective PI3Kα inhibition in combination with other agents for the treatment of PIK3CA-mutant tumors.
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http://dx.doi.org/10.1200/JCO.2017.72.7107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920739PMC
May 2018

Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function.

Sci Rep 2017 03 10;7:43873. Epub 2017 Mar 10.

Glycostem Therapeutics, Oss, The Netherlands.

Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCRγδ, CD19, CD14, SYTOX Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.
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http://dx.doi.org/10.1038/srep43873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345017PMC
March 2017

Robust 8-color flow cytometry panel reveals enhanced effector function of NKG2C CD57 FcεRγ NK cells in CMV seropositive human blood donors.

Immunobiology 2017 05 25;222(5):719-725. Epub 2017 Jan 25.

Immune Recovery Section, Department of Internal Medicine II, Division of Hematology, University Hospital of Würzburg, Würzburg, Germany.

Increasing evidence suggests that human NK cells may develop memory-like features. Here, we report the establishment of a robust 8-color flow cytometry panel that allows quantification and functional analysis of different memory-like NK cell subsets (NKG2C/CD57, FcεRγ) from relatively small blood samples. We could confirm previous publications reporting an enhanced prevalence of the mentioned memory-like NK cell subsets in CMV seropositive human donors and were able to show a clear congruence between enhanced expression of NKG2C and CD57, the absence of FcεRγ and CMV seropositivity supporting the hypothesis of memory-like NK cell development following viral infections. While we could not detect significant differences in effector functions (i.e. degranulation and production of IFNγ) in regard to age or CMV seropositivity when looking at the overall NK cell population, a significantly enhanced expression of CD107a and IFNγ could be observed in NKG2C/CD57 as well as FcεRγ NK cell subpopulations in CMV donors. This enhancement of effector functions was even more pronounced in NKG2C/CD57 NK cells that were also negative for FcεRγ; CMV seropositive donors showed a dramatically increased expression of CD107a as well as IFNγ. With only small-sized volumes of blood needed, our proposed 8-color panel and experimental protocol offers easy handling and a reliable and reproducible option for implementation in accompanying clinical research, e.g. for evaluation of immunosuppressed patients suffering from autoimmune diseases or in allograft recipients.
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http://dx.doi.org/10.1016/j.imbio.2017.01.005DOI Listing
May 2017

Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

PLoS One 2015 2;10(4):e0122689. Epub 2015 Apr 2.

Department of Internal Medicine II, Division of Hematology and Oncology, University Hospital of Würzburg, Würzburg, Germany.

Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122689PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383545PMC
April 2016

Phase 1/1B trial of the heat shock protein 90 inhibitor NVP-AUY922 as monotherapy or in combination with bortezomib in patients with relapsed or refractory multiple myeloma.

Cancer 2015 Jul 24;121(13):2185-92. Epub 2015 Mar 24.

Department of Hematology, Houston Methodist Cancer Center, Houston, Texas, USA.

Background: NVP-AUY922 (AUY; Luminespib) with or without bortezomib showed preclinical activity against multiple myeloma (MM) cells. This phase 1/1B study assessed NVP-AUY922 alone and with bortezomib in patients with relapsed or refractory MM.

Methods: Dose escalation was guided by an adaptive Bayesian logistic regression model. In phase 1, patients who progressed after 2 to 4 prior therapies received NVP-AUY922 intravenously once weekly. In phase 1B, patients who progressed after 2 or fewer prior therapies received NVP-AUY922 plus bortezomib. The primary objective was to determine the maximum tolerated dose (MTD) of NVP-AUY922.

Results: Twenty-four patients received NVP-AUY922 monotherapy at doses of 8 to 70 mg/m(2) . One dose-limiting toxicity (DLT) was observed (grade 3 blurred vision at 70 mg/m(2) ); no MTD was reached. The recommended phase 2 dose was 70 mg/m(2) . The most frequent drug-related adverse events (AEs) were diarrhea, nausea, and ocular toxicities. Grade 3/4 AEs were uncommon (<10%). Eight patients discontinued treatment because of AEs; 5 had ocular toxicities (≥45 mg/m(2) ). The best response was stable disease in 66.7% of the patients. There were no partial or complete responses. Five patients received NVP-AUY922 (which was started at 50 mg/m(2) ) plus bortezomib (1.3 mg/m(2) ). Three of these patients experienced DLT. No further dose escalation was performed; the MTD for NVP-AUY922 plus bortezomib was not established.

Conclusions: This study showed disease stabilization with NVP-AUY922 in patients with relapsed or refractory MM. The MTD for NVP-AUY922 was not reached, but reversible ocular toxicity has been reported at high dose levels. Bortezomib at the standard recommended dose plus NVP-AUY922 was not tolerated.
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http://dx.doi.org/10.1002/cncr.29339DOI Listing
July 2015

Dasatinib enhances migration of monocyte-derived dendritic cells by reducing phosphorylation of inhibitory immune receptors Siglec-9 and Siglec-3.

Exp Hematol 2014 Sep 29;42(9):773-82.e1-3. Epub 2014 May 29.

Comprehensive Cancer Center Mainfranken (CCC MF), University Hospital of Würzburg, Würzburg, Germany. Electronic address:

The SRC family of kinases (SFKs) is crucial to malignant growth, but also important for signaling in immune cells such as dendritic cells (DCs). These specialized antigen-presenting cells are essential for inducing and boosting specific T-cell responses against pathogens and malignancies. Targeted therapy with SFK inhibitors holds great promise as a direct anti-cancer treatment, but potentially also as an indirect treatment via immunomodulation. Here, we investigated whether the BCR-ABL/SRC inhibitor dasatinib would modulate the major effector functions of DCs, especially their migration, a prerequisite to interaction with lymphocytes in secondary lymphoid organs. We report for the first time that dasatinib more than doubled the number of mature human monocyte-derived DCs (moDCs) migrating toward a CCL19 gradient despite unchanged CCR7 expression when used for pretreatment. These effects were caused by dephosphorylation of SFKs, as confirmed by the specific SFK inhibitor SRC inhibitor 1, leading to dephosphorylation of the inhibitory immunoreceptors Siglec-9 and Siglec-3. The specific blocking of the latter also enhanced migration and underlined the importance of these SFK-dependent receptor systems for migration of moDCs. Dasatinib hampered the secretion of interleukin-12 by moDCs at clinically relevant concentrations. In contrast, endocytosis or boosting of cytomegalovirus-specific CD8(+) T-cell responses remained unaltered when applying dasatinib-pretreated moDCs, in line with minor effects on the expression of co-stimulatory molecules essential for DC-T cell interaction. The induction of enhanced migration of moDCs may potentially be useful in chemo-immunotherapeutic applications. Thus, the use of dasatinib or blocking Siglec antibodies as adjuvants in this setting to induce stronger immune responses is worthy of further study.
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http://dx.doi.org/10.1016/j.exphem.2014.05.010DOI Listing
September 2014

Modulation of natural killer cell effector functions through lenalidomide/dasatinib and their combined effects against multiple myeloma cells.

Leuk Lymphoma 2014 Jan 3;55(1):168-76. Epub 2013 Jun 3.

Immune Recovery Section, Division of Hematology.

The multikinase inhibitor dasatinib blocks the constitutive activation of oncogenic Src kinases in multiple myeloma (MM) cells and potentially enhances natural killer (NK) cell activity. Therefore, we tested combination effects of dasatinib and lenalidomide regarding MM cell viability and NK cell effector functions. The drug combination mostly had little influence on the viability of MM cell lines, and produced mixed results on primary MM cells. Prolonged lenalidomide treatment enhanced NK cell effector functions, and dasatinib addition at late stages of NK cell expansion increased levels of CD107a/b and interferon-γ (IFNγ), but not of tumor necrosis factor-α (TNFα). Additive effects were observed for the enhancement of cytokine production and degranulation, but only lenalidomide increased NK cell cytotoxicity against MM cells. This effect correlated with increased TNF-related apoptosis-inducing ligand (TRAIL) expression and was attenuated by dasatinib, or suppressors of TRAIL or TNFα. Our data thus indicate a functional role for the TRAIL/TRAIL-R system in lenalidomide-mediated NK-cell activity against MM cells, but also show that dasatinib is unsuitable to support or boost this effect.
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http://dx.doi.org/10.3109/10428194.2013.794270DOI Listing
January 2014

Combining dasatinib with dexamethasone long-term leads to maintenance of antiviral and antileukemia specific cytotoxic T cell responses in vitro.

Exp Hematol 2013 Jul 4;41(7):604-614.e4. Epub 2013 Mar 4.

Immune Recovery Section, Department of Internal Medicine II, Division of Hematology, Comprehensive Cancer Center Mainfranken, University Hospital of Würzburg, Germany.

Maintaining graft versus leukemia (GvL) and antivirus responses of cytotoxic T cells (CTLs) while suppressing graft-versus-host disease (GvHD) remains a challenge after allogeneic bone marrow transplantation. Clinical observations indicate that combining glucocorticoids with multi-tyrosine-kinase inhibitors could be a successful therapeutic approach. We and others have shown that the BCR-ABL/SRC kinase inhibitor dasatinib may enhance or suppress T cells in vitro. In this report, we evaluated combination effects of dasatinib and dexamethasone on CD3⁺ and virus-specific CD8⁺ T cells directly ex vivo and on antigen-specific leukemia-reactive and alloreactive CD8⁺ T cell clones. Functional outcomes assessed included cytokine production (IL-2, IFN-γ, TNF-α), degranulation (CD107a/b), activation (CD69 upregulation), proliferation, apoptosis and necrosis induction, and signal transduction. Overall, helper CD4⁺ T cells were more sensitive to inhibitory effects of the drug combination than cytotoxic CD8⁺ T cells and were more naive than memory T cell subsets. Of note, synergistic inhibitory effects occurred in different memory but not in naive T cell subsets. The drug combination inhibited virus-specific CD8⁺ T cell proliferation, but left cytokine production and degranulation unaltered, which may be due to the viral memory subset composition. Dasatinib rather hampered IFN-γ secretion and cytotoxic activity of human leukocyte antigen (HLA)-reactive CTLs, whereas effector functions of leukemia-reactive CTLs were maintained or enhanced when applied long term. Our data suggest that dasatinib might modulate GvL- differently than GvHD-promoting CTLs and provide a rationale to explore the drug combination further to treat GvHD while preserving GvL and antiviral CTL responses.
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http://dx.doi.org/10.1016/j.exphem.2013.02.013DOI Listing
July 2013

Killer-cell immunoglobulin-like receptor gene profile predicts good molecular response to dasatinib therapy in chronic myeloid leukemia.

Exp Hematol 2012 Nov 25;40(11):906-913.e1. Epub 2012 Jul 25.

Hematology Research Unit Helsinki, Department of Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland.

Tyrosine kinase inhibitors have greatly improved the prognosis of chronic myeloid leukemia (CML). In addition to direct kinase inhibition, their effects can also be mediated through immune modulation, such as expansion of cytotoxic T and natural-killer cells observed during dasatinib therapy. As natural-killer cell and partially CD8(+) T-cell function are regulated by killer immunoglobulin-like receptors (KIRs), we studied whether the KIR gene profile is associated with clinical therapy response in dasatinib-treated CML patients (n = 191). In first-line patients, the absence of the inhibitory KIR2DL5A (p = 0.0489), 2DL5B (p = 0.030), and 2DL5all (p = 0.0272) genes were associated with improved molecular response at the 12-month time point. In addition, the same trend was seen with two activating KIR genes, 2DS1 (p = 0.061) and 2DS2 (p = 0.071). Furthermore, when patients were clustered into two groups by their KIR gene profile, the BCR-ABL1 transcript levels differed significantly between the groups (p = 0.047), showing that patients who lacked several KIR genes had better response. The comparison of first-line and second-line patients did not show any significant differences in either KIR or human leukocyte antigen genotypes. Our results show that immunogenetic factors, such as the KIR gene profile, can play a role in tyrosine kinase inhibitor therapy response. Additional studies are warranted to elucidate the functional significance of KIR genes associated with treatment outcomes.
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http://dx.doi.org/10.1016/j.exphem.2012.07.007DOI Listing
November 2012

Enhancement of natural killer cell effector functions against selected lymphoma and leukemia cell lines by dasatinib.

Int J Cancer 2012 Sep 4;131(6):E916-27. Epub 2012 Apr 4.

Immune Recovery Section, Comprehensive Cancer Center, Department of Internal Medicine II, University of Würzburg, Würzburg, Germany.

As NK cell immunotherapy is still poorly successful, combinations with drugs enhancing NK cell activity are of major interest. NK large granular lymphocyte expansions associated with improved survival have been described under monotherapy with the Bcr-Abl/Src inhibitor dasatinib, which inhibits NK cell functions in vitro. As Src kinases play a major role in inhibitory and activating signaling pathways of NK cells, both outcomes appear plausible. To clarify these contradictory observations and potentially enable the use of dasatinib as adjuvant, we analyzed how clinically relevant doses promote NK cell effector functions. Polyclonal human NK cells were studied ex vivo. Functional outcomes assessed included conjugate formation, calcium flux, receptor regulation, cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction. While dasatinib inhibits NK cell effector functions during functional assays, 24 hr pretreatment of NK cells followed by washout of dasatinib, led to dose-dependent enhancement of cytokine production, degranulation marker expression and cytotoxicity against selected lymphoma and leukemia cell lines. Mechanistically, this was neither due to an altered viability of NK cells nor increased NKG2D, LFA-1 or conjugate formation with target cells. Receptor proximal signaling events were inhibited. However, a slight time dependent enhancement of Vav phosphorylation was observed under certain circumstances. The shift in Vav phosphorylation level may be one major mechanism for NK cell activity enhancement induced by dasatinib. Our findings argue for a careful timing and dosing of dasatinib application during leukemia/lymphoma treatment to enhance NK cell immunotherapeutic efforts.
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http://dx.doi.org/10.1002/ijc.27537DOI Listing
September 2012
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