Efficacy and safety of avelumab treatment in patients with metastatic Merkel cell carcinoma: experience from a global expanded access program

BackgroundAvelumab, a human anti–programmed death-ligand 1 immunoglobulin G1 monoclonal antibody, showed favorable efficacy and safety in patients with metastatic Merkel cell carcinoma (mMCC) in the phase II JAVELIN Merkel 200 trial, leading to approval in multiple countries. We describe real-world experience with avelumab in patients with mMCC from an expanded access program.MethodsEligible patients had mMCC and progressive disease during or after chemotherapy or were ineligible for chemotherapy or clinical trial participation. Patients received an initial 3-month supply of avelumab (administered as 10 mg/kg intravenously every 2 weeks until progressive disease or unacceptable toxicity); resupply was allowed following complete response, partial response, stable disease, or clinical benefit per physician assessment.ResultsBetween December 15, 2015, and March 4, 2019, 558 of 620 requests from 38 countries were medically approved, and 494 patients received avelumab. Among 240 evaluable patients, the objective response rate was 46.7% (complete response in 22.9%, including 3 of 16 potentially immunocompromised patients), and the disease control rate was 71.2%. The median duration of treatment in evaluable patients with response was 7.9 months (range, 1.0–41.7) overall and 5.2 months (range, 3.0–13.9) in immunocompromised patients. No new safety signals were identified. The expanded access program closed for new requests on December 31, 2018, as required after regulatory approval; benefitting patients continued to receive avelumab.ConclusionsThe avelumab expanded access program for patients with mMCC demonstrated efficacy and safety in a real-world setting, consistent with the results from JAVELIN Merkel 200, and provided a treatment for patients with limited options

Functional characterization of the serum and glucocorticoid inducible proteinkinase Sgk1

Titel und Inhaltsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Literatur Abkürzungen und Synonyme Zusammenfassung SummaryDie Serum- und Glukokortikoid-induzierbare Proteinkinase Sgk1 wird mit einer Vielzahl von Funktio-nen in verschiedenen Geweben und zellulären Kompartimenten in Verbindung gebracht. Sie soll eine Rolle bei der Steuerung zahlreicher Plasmamembrantransporter und Ionenkanäle sowie bei der Regu-lation des Zellzyklus spielen und an der Pathogenese einiger Erkrankungen beteiligt sein. Im Zentral-nervensystem wurde Sgk1 als aktivitätsreguliertes Gen im Hippocampus beschrieben, dessen Ex-pression sowohl nach neuronaler Aktivität als auch durch Stress induziert wird. Dabei führt neuronale Aktivität v.a. zur Expression in Neuronen des Gyrus dentatus während Stress zu einer Hochregulation in Oligodendrocyten verschiedener Fasertrakte führt. Um die teilweise widersprüchlichen Daten zur subzellulären Lokalisation von Sgk1 aufzuklären, wurde die Lokalisation der Kinase in dieser Arbeit durch konfokale Mikroskopie von Sgk1-EGFP-Fusionsproteinen untersucht. Dabei zeigte sich, daß Sgk1 hautsächlich und stimulusunabhängig an den Mitochondrien lokalisiert ist. In vitro-Import in aufgereinigte Mitochondrien sowie subzelluläre Fraktionierungen von Mauslebern bestätigten die mitochondriale Lokalisation von Sgk1. Durch bio-chemische Experimente konnte demonstriert werden, daß der N-Terminus von Sgk1 eine sehr starke Assoziation mit der äußeren Mitochondrienmembran vermittelt und daß sich der funktionell relevante Teil der Kinase auf der cytosolischen Seite der äußeren Membran befindet. Mit Hilfe von EGFP-fusionierten Deletionsvarianten konnte der mitochondriale Zielsteuerungsbereich von Sgk1 auf die Aminosäurereste 17-33 eingegrenzt werden. Pulse-chase Experimente zeigten, daß der gleiche Se-quenzbereich auch für die kurze Halbwertzeit von Sgk1 verantwortlich ist, so daß der schnelle Abbau und die subzelluläre Lokalisation von Sgk1 untrennbar miteinander gekoppelt sind. Die Befunde dieser Arbeit deuten darauf hin, daß die Kinase nach Erreichen ihrer mitochondrialen Destination stabilisiert wird, und der Abbau hauptsächlich im Cytosol stattfindet. NDRG1 ist das einzige Protein, von dem zweifellos gezeigt wurde, daß es ausschließlich durch Sgk1 phosphoryliert wird. In dieser Arbeit konnte gezeigt werden, daß die Effizienz zur Phosphorylierung von NDRG1 durch die mitochondriale Lokalisation von Sgk1 erhöht wird. Darüberhinaus wurde NDRG1 als Sgk1-Phosphorylierungstarget im Hippocampus der Maus nach neuronaler Aktivität und Gabe von Glukokortikoiden identifiziert. Bei der Analyse der räumlichen mRNA-Expression von Sgk1 im Gehirn der Maus zeigte sich, daß sich das Expressionmuster von Sgk1 nach der Applikation von Dexamethason kaum von dem bei Kontrolltieren unterscheidet. Dieser Befund weist darauf hin, daß Sgk1 durch Glukokortikoidrezeptor- Agonisten aktiviert wird, und daß hauptsächlich Katecholamine für die Stress- induzierte Sgk1-Expression in Oligodendrocyten verantwortlich sind. Darüberhinaus macht es der Vergleich mit dem Expressionsmuster von NDRG1 wahrscheinlich, daß NDRG1 hauptsächlich in den Nervenzellen des Hippocampus durch Sgk1 phosphoryliert wird. Bei der Suche nach einer zellulären Funktion von Sgk1 zeigte sich, daß die Kinase weder einen Einfluß auf Teilung oder Fusion von Mitochondrien, noch auf die Steuerung der Genexpression in HEK293 Zellen hat, da sich nach Überexpression von Sgk1 in dieser Zellinie keine Unterschiede in der Mitochondrienmorphologie oder im Transkriptom fanden. Weder mit einem im Rahmen dieser Arbeit hergestellten Antikörper, noch mit vier weiteren Antikör-pern gelang die Detektion von Sgk1 in primärem Mausgewebe oder nicht transfizierten Zellinien, was auf die sehr kurze Halbwertzeit von Sgk1 zurückzuführen ist. Erst nach Applikation von Dexametha- son bei Tieren oder starker Überexpression in HEK293 Zellen ließ sich Sgk1 in Western Blot Analysen detektieren. In HEK293 Zellen trat dabei eine zusätzliche Bande bei 40 kD auf, von der gezeigt werden konnte, daß sie durch Benutzung eines alternativen Translationsstarts entsteht. Die Ergebnisse dieser Arbeit stellen viele publizierte Daten zu Sgk1 in Frage, insbesondere im Hin-blick auf die subzelluläre Lokalisation, bestimmte zelluläre Funktionen und Expressionsanalysen mit Hilfe von Sgk1-spezifischen Antikörpern. Sie können aber als Ausgangspunkt weiterer Experimente zu Aufklärung der zellulären Funktion von Sgk1 dienen.The serum- and glucocorticoid-inducible proteinkinase Sgk1 has been implicated in a variety of functions in different tissues and subcellular compartments. It has been proposed that the kinase regulates the activity of plasma membrane transporters and ion channels, has an influence on the cell cycle and might have a role in the pathogenesis of different diseases. Sgk1 has been identified as an activity regulated gene in the rodent hippocampus. The expression of Sgk1 mRNA is upregulated in hippocampal neurones after neuronal activity and in oligodendrocytes in the white matter after general stress. To clarify the inconsistent data concerning the subcellular localization of Sgk1, HEK293 cells were transfected with Sgk1-EGFP and analyzed by confocal microscopy. The fusion protein was mainly localized to the mitochondria irrespective of any stimuli that have been reported to influence the subcellular localization of the kinase. In vitro import assays using purified mitochondria and subcellular fractionations of mouse liver tissue confirmed the predominant mitochondrial localization of Sgk1. Biochemical experiments demonstrated that the N-terminus of Sgk1 confers a strong association with the outer mitochondrial membrane and that the kinase is localized on the cytosolic surface of the organelles. Microscopy of EGFP-tagged deletion variants identified amino acids 17-33 as the mitochondrial targeting sequence of Sgk1. Pulse-chase experiments demonstrate that the identical residues also determine the short half-life of the Sgk1 protein. This makes it likely that the kinase is stabilized upon arrival at its mitochondrial destination and degradation mainly occurs in the cytosol. NDRG1 is the only known unambiguous phosphorylation target of Sgk1. Here it could be shown that Sgk1 mediated phosphorylation of NDRG1 is more efficiant when Sgk1 is localizated to mitochondria. Moreover, NDRG1 has been identified as a substrate for Sgk1 in the mouse hippocampus after neuronal activity and after application of the glucocorticoid receptor agonist dexamethasone. Analysis of the spatial mRNA expression pattern of Sgk1 in the mouse brain revealed only minor differences between dexamethasone treated mice and control animals. This makes it likely that agonists of the glucocorticoid receptor can activate the kinase and that catecholamines are responsible for the stress induced expression of Sgk1 in oligodendrocytes. Comparison of the expression patterns of Sgk1 and NDRG1 render it obvious that NDRG1 is a phosphorylation target of Sgk1 in the granule cells and pyramidal cells of the hippocampus. Attempts to identify a cellular function for Sgk1 disclosed no role for Sgk1 in the fission or fusion of mitochondria nor in the regulation of gene expression, as overexpression of Sgk1 in HEK293 cells had no influence on the morphology of mitochondria or the transcriptome. Using five different antibodies including one that has been generated during this thesis it was not possible to detect the Sgk1 protein in primary mouse tissues or untransfected cell lines. Only after the administration of dexamethasone in animals, or after strong overexpression in HEK293 cells Sgk1 could be detected in Western Blot analysis. Thereby in HEK293 cells a second specific band at approximatly 40 kD occurred. It could be demonstrated that this second species originates from the usage of an alternative translational start. The results of this thesis challange the data of several publications, especially with respect to subcellular localization and postulated cellular functions of Sgk1. Moreover, they cast any antibody- dependent expression analysis of Sgk1 into doubt. On the other hand, the presented data may give rise to further substantiated analysis that shed more light on the cellular function of Sgk1

Efficacy and safety of avelumab treatment in patients with metastatic Merkel cell carcinoma: experience from a global expanded access program

BackgroundAvelumab, a human anti–programmed death-ligand 1 immunoglobulin G1 monoclonal antibody, showed favorable efficacy and safety in patients with metastatic Merkel cell carcinoma (mMCC) in the phase II JAVELIN Merkel 200 trial, leading to approval in multiple countries. We describe real-world experience with avelumab in patients with mMCC from an expanded access program.MethodsEligible patients had mMCC and progressive disease during or after chemotherapy or were ineligible for chemotherapy or clinical trial participation. Patients received an initial 3-month supply of avelumab (administered as 10 mg/kg intravenously every 2 weeks until progressive disease or unacceptable toxicity); resupply was allowed following complete response, partial response, stable disease, or clinical benefit per physician assessment.ResultsBetween December 15, 2015, and March 4, 2019, 558 of 620 requests from 38 countries were medically approved, and 494 patients received avelumab. Among 240 evaluable patients, the objective response rate was 46.7% (complete response in 22.9%, including 3 of 16 potentially immunocompromised patients), and the disease control rate was 71.2%. The median duration of treatment in evaluable patients with response was 7.9 months (range, 1.0–41.7) overall and 5.2 months (range, 3.0–13.9) in immunocompromised patients. No new safety signals were identified. The expanded access program closed for new requests on December 31, 2018, as required after regulatory approval; benefitting patients continued to receive avelumab.ConclusionsThe avelumab expanded access program for patients with mMCC demonstrated efficacy and safety in a real-world setting, consistent with the results from JAVELIN Merkel 200, and provided a treatment for patients with limited options

Human Cytomegalovirus Gene Products US2 and US11 Differ in Their Ability To Attack Major Histocompatibility Class I Heavy Chains in Dendritic Cells

Human cytomegalovirus (HCMV) encodes several proteins that inhibit major histocompatibility complex (MHC) class I-dependent antigen presentation. The HCMV products US2 and US11 are each sufficient for causing the dislocation of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol, where the heavy chains are readily degraded. The apparent redundancy of US2 and US11 has been probed predominantly in cultured cell lines, where differences in their specificities were shown for murine and human MHC class I locus products. Here, we expressed US11 and US2 via adenovirus vectors and show that US11 exhibits a superior ability to degrade MHC class I molecules in primary human dendritic cells. MHC class II complexes are unaffected by US2- and US11-mediated attack. We suggest that multiple HCMV-encoded immunoevasions have evolved complementary functions in response to diverse host cell types and tissues

EBAG9 Adds a New Layer of Control on Large Dense-Core Vesicle Exocytosis via Interaction with Snapin

Regulated exocytosis is subject to several modulatory steps that include phosphorylation events and transient protein–protein interactions. The estrogen receptor-binding fragment-associated gene9 (EBAG9) gene product was recently identified as a modulator of tumor-associated O-linked glycan expression in nonneuronal cells; however, this molecule is expressed physiologically in essentially all mammalian tissues. Particular interest has developed toward this molecule because in some human tumor entities high expression levels correlated with clinical prognosis. To gain insight into the cellular function of EBAG9, we scored for interaction partners by using the yeast two-hybrid system. Here, we demonstrate that EBAG9 interacts with Snapin, which is likely to be a modulator of Synaptotagmin-associated regulated exocytosis. Strengthening of this interaction inhibited regulated secretion of neuropeptide Y from PC12 cells, whereas evoked neurotransmitter release from hippocampal neurons remained unaltered. Mechanistically, EBAG9 decreased phosphorylation of Snapin; subsequently, association of Snapin with synaptosome-associated protein of 25 kDa (SNAP25) and SNAP23 was diminished. We suggest that the occurrence of SNAP23, Snapin, and EBAG9 also in nonneuronal cells might extend the modulatory role of EBAG9 to a broad range of secretory cells. The conjunction between EBAG9 and Snapin adds an additional layer of control on exocytosis processes; in addition, mechanistic evidence is provided that inhibition of phosphorylation has a regulatory function in exocytosis

Management of crizotinib therapy for ALK-rearranged non-small cell lung carcinoma: An expert consensus

Within 4 years of the discovery of anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC), the ALK inhibitor crizotinib gained US and European approval for the treatment of advanced ALK-positive NSCLC. This was due to the striking response data observed with crizotinib in phase I and II trials in patients with ALIC-positive NSCLC, as well as the favorable tolerability and safety profile observed. Recently published phase III data established crizotinib as a new standard of care for this NSCLC molecular subset. A consequence of such rapid approval, however, is the limited clinical experience and relative paucity of information concerning optimal therapy management. In this review, we discuss the development of crizotinib and the clinical relevance of its safety profile, examining crizotinib-associated adverse events in detail and making specific management recommendations. Crizotinib-associated adverse events were mostly mild to moderate in severity in clinical studies, and appropriate monitoring and supportive therapies are considered effective in avoiding the need for dose interruption or reduction in most cases. Therapy management of patients following disease progression on crizotinib is also discussed. Based on available clinical data, it is evident that patients may have prolonged benefit from crizotinib after Response Evaluation Criteria in Solid Tumors-defined disease progression, and crizotinib should be continued for as long as the patient derives benefit. (C) 2014 The Authors. Published by Elsevier Ireland Ltd