Acute Motor Axonal Neuropathy in a Patient with Metastatic Pancreatic Neuroendocrine Tumor Receiving Chemotherapy with Capecitabine and Temozolomide: A Case Report

We report a case of a 37- year old female patient with metastatic pancreatic neuroendocrine tumor that developed acute motor axonal neuropathy after receiving chemotherapy with capecitabine and temozolomide. She had repeatedly progressed after several surgical resections of her liver metastases, other hepatic directed procedures, several sessions of peptide receptor radionuclide therapy and two systemic treatment lines. When we started a third line therapy with capecitabine (1500 mg absolute twice daily, day 1-14) and temozolomide (360 mg absolute in two divided doses on day 1-5) in a 28 days long cycle, she unexpectedly developed a progressive weakness of the upper and lower limbs and dysphagia. The diagnosis of acute motor axonal neuropathy was confirmed by nerve conduction studies. The patient`s condition improved rapidly after chemotherapy was stopped and plasma exchange (nine sessions) as well as a maintenance therapy with intravenous immunoglobulins was started. She was almost asymptomatic seven months after onset of symptoms. This case describes a previously unreported association between acute motor axonal neuropathy and chemotherapy with capecitabine and temozolomide

Concomitant statin use does not impair the clinical outcome of patients with diffuse large B cell lymphoma treated with rituximab-CHOP

Preclinical data indicated a detrimental effect of statins on the anti-lymphoma activity of rituximab. We evaluated the impact of concomitant statin medication on the response and survival of patients with diffuse large B cell lymphoma (DLBCL) receiving rituximab-cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) as first-line therapy. Medical histories of patients with DLBCL who were treated with R-CHOP as first-line therapy were assessed for concomitant statin use, response after completion of chemotherapy, event-free survival (EFS), and overall survival (OS). Furthermore, 2-[18F]fluor-2-deoxyglucose (FDG)-PET/CT results after completion of first-line therapy were compared between the groups. Overall, 145 patients with DLBCL treated with R-CHOP from January 2001 to December 2009 were analyzed. Twenty-one (15%) patients received statins throughout therapy. Five-year EFS was 67.3% in patients without statins compared with 79% in patients receiving statins during R-CHOP (HR, 0.47; 95% CI, 0.15-1.54, p = 0.2). Five-year OS was 81.4% for patients without statins compared with 93.3% for patients taking statins (HR, 0.58; 95% CI 0.07-4.55, p = 0.6). There were no statistically significant differences in the rates of complete remissions between the two groups (75% in the non-statin group versus 86% in the statin group, p = 0.45). A trend toward a lower rate of complete metabolic responses in FDG-PET/CT after chemotherapy was seen in patients without statin medication compared with the patients taking statins (84% versus 92%, p = 0.068). Concomitant statin use had no adverse impact on response to chemotherapy, EFS, and OS in patients treated with R-CHOP for DLBC

Pegfilgrastim reduces the length of hospitalization and the time to engraftment in multiple myeloma patients treated with melphalan 200 and auto-SCT compared with filgrastim

To reduce the duration of neutropenia after conditioning chemotherapy and autologous peripheral blood stem cell transplantation (APBSCT), granulocyte-colony stimulating factors (G-CSF) are commonly administered. We retrospectively evaluated the impact of pegfilgrastim compared to filgrastim on neutrophil engraftment, hospital stay, and supportive measures in patients with multiple myeloma after conditioning with Melphalan 200 (Mel200) followed by APBSCT. Ninety-two APBSCT after Mel200 treatment were performed in 72 patients between January 2006 and December 2009 at our institution. Patients received either single-dose pegfilgrastim (n = 46; 50%), or daily filgrastim (n = 46; 50%) after APBSCT (median duration of filgrastim use, 9days; range, 3-14days). Duration of neutropenia grade IV was shorter with pegfilgrastim compared with filgrastim (median, 5days (range, 3-14days) versus 6days (range, 3-9days), p = 0.0079). The length of hospitalization differed significantly (pegfilgrastim (median, 14.5days; range, 11-47days) versus filgrastim (median, 15.5days; range, 12-64days), p = 0.024). Pegfilgrastim-treated patients had less red blood cell transfusions (median, 0 transfusions (range, 0-10) versus 0.5 transfusions (range, 0-9), p = 0.00065). Pegfilgrastim was associated with reduced cost of the treatment procedure compared with filgrastim (p = 0.031). Pegfilgrastim appears to be at least equivalent to filgrastim without additional expenditure in myeloma patients treated with Mel200 and APBSC

Noninvasive rapid detection of metabolic adaptation in activated human T lymphocytes by hyperpolarized 13 C magnetic resonance

Abstract: The metabolic shift induced in human CD4+ T lymphocytes by stimulation is characterized by an upregulation of glycolysis, leading to an augmentation in lactate production. This adaptation has already been highlighted with various techniques and reported in several previous studies. We herein propose a method to rapidly and noninvasively detect the associated increase in flux from pyruvate to lactate catalyzed by lactate dehydrogenase using hyperpolarized 13C magnetic resonance, a technique which can be used for in vivo imaging. It was shown that the conversion of hyperpolarized 13C-pyruvate to 13C-lactate during the one-minute measurement increased by a mean factor of 3.6 in T cells stimulated for 5 days as compared to resting T cells. This method can be extended to other metabolic substrates and is therefore a powerful tool to noninvasively analyze T cell metabolism, possibly in vivo

Aberrant Lck Signal via CD28 Costimulation Augments Antigen-Specific Functionality and Tumor Control by Redirected T Cells with PD-1 Blockade in Humanized Mice

Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed. Fibroblast activation protein (FAP)-specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized and antitumor efficacy was tested in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM. All the three CARs demonstrated FAP-specific functionality Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality. Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted

Establishment of an expression system in Pichia pastoris to produce recombinant fusion antibodies exemplarily used for the A33rbGFP fusion antibody.

Titelblatt, Inhaltsverzeichnis, Abkürzungen 1\. Einleitung 1 2\. Material und Methoden 22 3\. Ergebnisse 48 4\. Diskussion 83 5\. Zusammenfassung 102 6\. Literaturverzeichnis 104 Danksagung 117Fusionsantikörper sind bifunktionale, rekombinante Moleküle aus einem Antigen- bindenden Antikörperfragment und einem weiteren Molekül, zum Beispiel einem Enzym. Solche Fusionsantikörper werden im Wesentlichen für die Anwendung in der antineoplastischen Chemotherapie konzipiert. In diesem Zusammenhang ist die antikörpergesteuerte Enzym-Prodrug-Therapie (ADEPT) ein zweistufiges Behandlungskonzept, wobei im ersten Schritt der Fusionsantikörper an ein tumor- oder tumorgewebespezifisches Antigen bindet. Im zweiten Schritt wandelt ein mit diesem Antikörper fusioniertes Enzym eine systemisch applizierte, ungiftige Arzneimittelvorstufe (Prodrug) im Tumorgewebe in ein aktives Chemotherapeutikum um, wodurch dessen zytotoxische Wirkung auf den Tumor fokussiert wird. Aufgrund der bisher unbefriedigenden Ergebnisse der Behandlung des kolorektalen Karzinoms stellt die antikörpergesteuerte Enzym- Prodrug-Therapie eine möglicherweise verbesserte adjuvante oder palliative Therapiestrategie dar. Das A33-Antigen wird von 95% der Kolonkarzinome präsentiert und kann genutzt werden, um sowohl Primärtumoren wie Metastasen mit hoher Sensitivität zu detektieren. Es bietet sich damit als Zielantigen einer antikörpergesteuerten Enzym-Prodrug-Therapie an. Um den experimentellen oder klinischen Einsatz von Fusionsantikörpern für ADEPT zu realisieren, sind Expressionssysteme notwendig, die komplexe Proteine in löslicher und funktionaler Form in größerem Maßstab exprimieren können. Mit den vorgestellten Ergebnissen wurde ein solches Expressionssystem zur Herstellung von Fusionsantikörpern in Hefen des Stammes Pichia pastoris etabliert. Dabei wurde beispielhaft der Fusionsantikörper A33rbGFP exprimiert. Er besteht aus dem gegen das A33-Antigen gerichtete A33-Single-Chain-Fragment und einem Green-Fluorescent-Protein (GFP). Das Green-Fluorescent-Protein wurde für dieses Pilotprojekt gewählt, da mit diesem Fusionsprotein einschrittige Fluoreszenzanalysen zum Bindungsverhalten des Fusionsantikörpers durchführbar waren. Das spezifische Bindungsverhalten des Konstrukts an verschiedenen Kolonkarzinomzelllinien konnte vergleichend gezeigt werden. Darüber hinaus wurde versucht, den A33rbCD Fusionsantikörper zu exprimieren, bei dem das Green-Fluorescent-Protein gegen eine bakterielle Cytosindeaminase (CD) als Effektorenzym für ADEPT ausgetauscht wurde. Die Cytosindeaminase katalysiert die Umwandlung der ungiftigen Arzneimittelvorstufe 5?Fluorcytosin in die zytotoxische Wirksubstanz 5-Fluoruracil. Dabei war festzustellen, dass die DNA-Sequenz dieses Fusionsantikörpers in der vorliegenden Form nicht im Hefesystem zu exprimieren war. Es wurde deshalb ein analoges Fusionskonstrukt entworfen und kloniert, in dem anstelle des bakteriellen Enzyms die aus der Hefe Saccharomyces cerevisiae stammende Cytosindeaminase (CDy) verwendet wird. Auf diese Weise soll eine Expression dieses Fusionsantikörpers für ADEPT in der Hefe Pichia pastoris ermöglicht werden.Fusion antibodies are recombinant constructs comprising an antigen-binding antibody fragment and an effector molecule such as an enzyme. Such constructs have mainly been designed for use in is antineoplastic therapy. In this context, antibody-directed enzyme-prodrug therapy (ADEPT) is a two-step approach utilizing fusion-antibodies for the tumor-specific activation of inert prodrugs. In the first step, the antibody construct binds to a tumor- specific antigen. Subsequently, the enzyme component of the construct converts a systemically applied, relatively non-toxic prodrug into a cytotoxic metabolite, thus focusing its activity to the tumor tissue. Given the hitherto unsatisfactory outcome of chemotherapy for colorectal carcinoma, ADEPT may provide a means of improved adjuvant or palliative therapy for this cancer. The A33 antigen is expressed on the cell surface in 95% of colon carcinomas and has been utilized to detect both primary and metastatic tumor sites with high sensitivity. Thus, it is a potential target antigen for ADEPT. To realize the experimental or clinical use of fusion antibodies for ADEPT, expression systems are required which can produce in large scale complex proteins in soluble and functional form. The results presented here establish such an expression system for the production of fusion antibodies in the yeast Pichia pastoris, using an A33rbGFP fusion protein as a pilot subject. This fusion protein consisted of a single chain variable region fragment (scFv) directed against the A33 antigen, termed A33rb, and green fluorescent protein (GFP). GFP has been chosen for this pilot project because it allows for one-step fluorescence analysis of fusion protein binding. Specific binding could be demonstrated, comparing various colon carcinoma cell lines quantitatively. In addition, attempts were made to express the fusion construct A33rbCD, which contains the bacterial enzyme cytosine deaminase (CD) instead of GFP. A potential effector enzyme for ADEPT, CD catalyses the conversion of 5-fluorocytosine, which is non-toxic in humans, into the cytotoxic drug 5-fluorouracil. The results of extensive experiments, however, suggested that this fusion construct in the selected form could not be expressed in the yeast system. Therefore we designed and cloned an analogous construct, replacing the bacterial CD enzyme with the isoenzyme from the yeast Sacharomyces cerevisiae (CDy). This new construct is expected to be produced more readily in the yeast expression system

T Cell Engineering

T cells are a new and promising antigen-specific therapeutic option for the treatment of malignant diseases. To achieve antigen specificity against tumor antigens, T cells can be manipulated by gene transfer to express chimeric antigen receptors (CARs). CAR-expressing T cells are called redirected T cells. CARs are composed of an extracellular antibody-derived antigen recognition domain, a transmembrane domain and a cytoplasmatic signal domain. Therefore, redirected T cells combine the exchangeable specificity of an antibody with the cytotoxic machinery of a T cell. Early clinical trials with redirected T cells targeting cluster of differentiation (CD) 19 have shown impressive results in CD19-positive hematological cancers. However, for solid cancers only limited clinical experience exists and new and innovative concepts have to be developed to overcome tumor-mediated immune suppression. Herein, we describe the general design of a CAR, the function of the different domains and the different strategies to produce redirected T cells. Furthermore, we summarize and discuss the preclinical and clinical data indicating the tremendous potential of redirected T cells to become a mainstay of cancer immunotherapy

Is distinct EGFR mutation status proof of second simultaneous primary non-small cell lung cancer?

We present the case of a 54-year-old woman diagnosed with two simultaneous adenocarcinomas in the right and left upper lobe of the lungs. The patient was treated with two curative intended resections and chemotherapy. The molecular analysis showed distinct EGFR mutation pattern in both adenocarcinomas of the lung. During the clinical course, the adenocarcinoma without EGFR mutation relapsed multiple times in the right lung resulting finally in pneumonectomy in 2009. Since then, the patient had no indication of relapse. The case demonstrates the difficulty to clinically discriminate a palliative metastatic situation from a potential curable situation with two simultaneous primary adenocarcinomas of the lung. We propose that a different molecular pattern between lesions could be used as an argument for the presence of two simultaneous primary tumors. In the case of distinct EGFR mutation pattern, therapy could be performed with curative intent