Targeted ultra-deep sequencing reveals recurrent and mutually exclusive mutations of cancer genes in blastic plasmacytoid dendritic cell neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare haematopoietic malignancy characterized by dismal prognosis and overall poor therapeutic response. Since the biology of BPDCN is barely understood, our study aims to shed light on the genetic make-up of these highly malignant tumors. Using targeted high-coverage massive parallel sequencing, we investigated 50 common cancer genes in 33 BPDCN samples. We detected point mutations in NRAS (27.3% of cases), ATM (21.2%), MET, KRAS, IDH2, KIT (9.1% each), APC and RB1 (6.1% each), as well as in VHL, BRAF, MLH1, TP53 and RET (3% each). Moreover, NRAS, KRAS and ATM mutations were found to be mutually exclusive and we observed recurrent mutations in NRAS, IDH2, APC and ATM. CDKN2A deletions were detected in 27.3% of the cases followed by deletions of RB1 (9.1%), PTEN and TP53 (3% each). The mutual exclusive distribution of some mutations may point to different subgroups of BPDCN whose biological significance remains to be explored

Identifizierung und Charakterisierung T-zellerkannter Tumorantigene im Melanommodell MZ7

Die Kenntnis immunogener Tumorantigene ist Grundlage für die rationale Entwicklung immunologisch orientierter Therapieverfahren. In der vorliegenden Arbeit wurden im autologen Melanommodell MZ7 drei neue T-zellerkannte Tumorantigene vorgestellt. Während nahezu alle bisher beschriebenen Tumorantigene mit Hilfe von T-Zellklonen (CTL) entdeckt wurden, die aus tumorreaktiven MLTCs (gemischte Lymphozyten/Tumor- Zellkulturen) generiert worden waren, wurde in dieser Arbeit versucht, wenige Wochen stimulierte MLTCs direkt zur Antigensuche zu verwenden. Als sensitives Nachweissystem wurde der IFNg-ELISPOT-Assay eingesetzt. Die Motivation dazu waren einerseits praktische Erwägungen, da CTL-Klonierungen material- und zeitaufwendig sind. Andererseits wurde vermutet, dass die Etablierung von CTL-Klonen in Langzeitkultur neben Zufallsprozessen auch Selektionsprozessen unterliegt, die CTL mit bestimmten Eigenschaften favorisieren. Eventuell eröffnete sich durch den Einsatz der MLTCs die Möglichkeit, Antigene zu entdecken, die mit Hilfe permanent kultivierter CTL aus den MLTCs nicht gefunden würden.Um beispielsweise Schwankungen im Proliferationsverhalten und in Effektorfunktionen permanent kultivierter T-Zellen zu umgehen, wurden für die Versuche in dieser Arbeit in Portionen eingefrorene T-Zellen verwendet, die zuvor zu ausreichender Menge expandiert worden waren. Es ließ sich zeigen, dass durch die Kryokonservierung der T- Zellen zu einem bestimmten Zeitpunkt nach einer Restimulation mit den Tumorzellen der Aktivierungszustand der T-Zellen konserviert wurde, und dass die T-Zellen nach dem Auftauen in ELISPOT-Assays ohne wesentliche Einbußen spezifisch auf einen erneuten Antigenkontakt reagierten. Bei der Testung von mehreren, unabhängig generierten MLTCs gegen bekannte Tumorantigene wurden T-Zellantworten gegen gp100/HLA-B7.2, Tyrosinase/HLA-A26.1, SIRT2P182L/HLA-A3.1 und, deutlich stärker, gegen die Melanomzelllinie festgestellt. Nachfolgend wurde mit Hilfe bereits etablierter CTL-Klone die peptidkodierende Region von gp100 über die Transfektion von gp100-Fragmenten in COS-7-Zellen eingegrenzt und anschließend ein synthetisches Peptid identifiziert, das von den CTL-Klonen erkannt wurde. Das zweite identifizierte Tumorantigen, Tyrosinase/HLA-A26.1, wurde nur in einer von sechs neu generierten, unabhängigen MLTCs entdeckt. Da keine Tyrosinase/HLA-A26.1-reaktiven CTL-Klone zur Verfügung standen, wurden die zur Eingrenzung der peptidkodierenden Region transfizierten COS-7-Zellen mit der MLTC selbst getestet. Anschließend wurde ein synthetisches Peptid identifiziert, das von der MLTC erkannt wurde.Die Reaktivität der MLTCs gegen die Melanomzelllinie war bei weitem nicht durch die bis dahin gefundenen T-Zellantworten erklärbar. Daher wurde mit einer der MLTCs versucht, durch cDNA-Expressionsklonierung ein neues Antigen in der cDNA-Bank aus dem MZ7-Melanom zu identifizieren. Die Reaktivität der MLTC gegen den Tumor war hauptsächlich durch einen Antikörper gegen HLA-A3 blockierbar. Daher wurde HLA-A*03011-cDNA für das „Screening“ kotransfiziert. Das Verfahren führte zur Identifizierung eines weiteren punktmutierten Tumorantigens: GPNMBG181D (GPNMBmutiert). Die Mutation führte dazu, dass ein Teil eines ungespleißten Introns Bestandteil der im Tumor entdeckten cDNA war. Ein aus der Exon/Intron-Region kodiertes synthetisches Peptid mit der ausgetauschten Aminosäure wurde von der MLTC deutlich erkannt. Durch RT-PCR-Analysen wurde im MZ7-Melanom, in fast allen getesteten weiteren Melanomen sowie in einem Teil der überprüften Nierenzellkarzinome eine Variante der GPNMB-cDNA entdeckt, in der ebenfalls das erwähnte Intron enthalten war, ohne dass die Mutation vorlag (GPNMB/INT4). Diese Spleißvariante wurde nicht in EBV-B-Zelllinien und anderen Tumorzelllinien gefunden. Zusätzlich zu dem bereits zuvor charakterisierten Tumorantigen SIRT2P182L wurden drei weitere T-zellerkannte Antigene im Melanommodell MZ7 identifiziert. T-Zellreaktivität gegen das Antigen GPNMBG181D entwickelte sich, im Gegensatz zu den anderen Antigenen, in allen getesteten MLTCs. Dies spricht für eine immunologische Dominanz des Antigens im Melanommodell MZ7. Die Tatsache, dass das „stärkste“ Antigen mit Hilfe einer MLTC identifiziert wurde, bietet die Aussicht, evtl. weitere dominante Antigene mit dem Verfahren identifizieren zu können. Die Verwendung von kurzzeitstimulierten MLTCs anstelle von permanent kultivierten CTL-Klonen stellt einen ersten Schritt auf dem Weg zur Beschleunigung der Suche nach Tumorantigenen dar. Die Verfahrensbeschleunigung ist die Voraussetzung dafür, in Zukunft Antigene nicht nur in archivierten Modellen zu suchen, sondern in Patienten zu einem frühen Zeitpunkt der malignen Erkrankung. Dann bestünde die Chance, dass die Kenntnis individueller Tumorantigene in immuntherapeutische Konzepte eingeht

How Soluble GARP Enhances TGFβ Activation.

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo

How soluble GARP enhances TGFβ activation

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo

HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model

T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient’s HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy

Exome sequencing to predict neoantigens in melanoma

The ability to use circulating peripheral blood cells and matched tumor sequencing data as a basis for neoantigen prediction has exciting possibilities for application in the personalized treatment of cancer patients. Wehave used a high-throughput screening approach, combining whole-exome sequence data, mRNA microarrays, and publicly available epitope prediction algorithm output to identify mutated proteins processed and displayed by patient tumors and recognized by circulating immune cells. Matched autologous melanoma cell lines and peripheral blood mononuclear cells were used to create mixed lymphocyte tumor cell cultures, resulting in an expansion of tumor-reactive T cells to use for mutated peptide screening. Five patients were investigated, three of whom had a durable complete response (CR; 15+ years) in an autologous melanoma-pulsed dendritic cell clinical trial. We identified seven mutated antigens in total that stimulated T-effector memory cells in two of the five patients. While the procedure did not result in clinically applicable neoantigens for all patients, those identified were likely important in tumor clearance, leading to durable CR. The nature of the screening process allows results to be obtained rapidly and is easily applicable to a wide variety of different tumor types. (C) 2015 AACR

Gamma Irradiation Triggers Immune Escape in Glioma-Propagating Cells

Glioblastoma multiforme is the most common and devastating form of brain tumor for which only palliative radio- and chemotherapy exists. Although some clinical studies on vaccination approaches have shown promising efficacy due to their potential to generate long-term immune surveillance against cancer cells, the evasion mechanisms preventing therapy response are largely uncharacterized. Here, we studied the response of glioblastoma-propagating cells (GPCs) to clinically relevant doses of γ radiation. GPCs were treated with 2.5 Gy of γ radiation in seven consecutive cellular passages to select for GPCs with increased colony-forming properties and intrinsic or radiation-induced resistance (rsGPCs). Quantitative proteomic analysis of the cellular signaling platforms of the detergent-resistant membranes (lipid rafts) in GPCs vs. rsGPCs revealed a downregulation of the MHC class I antigen-processing and -presentation machinery. Importantly, the radio-selected GPCs showed reduced susceptibility towards cytotoxic CD8+ T-cell-mediated killing. While previous studies suggested that high-dose irradiation results in enhanced antigen presentation, we demonstrated that clinically relevant sub-lethal fractionated irradiation results in reduced expression of components of the MHC class I antigen-processing and -presentation pathway leading to immune escape

<i>In vivo</i> coupling of GARP_TS to recombinant TGFβ.

<p>HEK 293H cells were transfected with plasmids containing the cDNA of the constructs GARP_FL, TGFβ_Strep or both in combination. 48h after transfection, the culture medium was exchanged for FCS-free DMEM supplemented with NEAA. Cell lysates were prepared using 200 μl RIPA buffer per 1x10⁶ cells. Samples were separated on a 10% PAA SDS-PAGE followed by western blotting on a PVDF membrane. For molecular size determination the magic mark XP marker (Invitrogen; Darmstadt, Germany) was used. For detection the blot was probed with anti-Strep-tag and anti-His-tag antibodies, respectively (Quiagen; Hilden, Germany). As secondary antibody a peroxidase coupled anti-mouse-IgG antibody (Dianova; Hamburg, Germany) was used.</p

<i>In vitro</i> coupling of GARP_TS to recombinant TGFβ.

<p>(A) 300 ng GARP_TS and 600 ng recombinant latent TGFβ per lane were incubated overnight in the presence or absence of a redox-buffer containing 0.05 mM GSSH and 2 mM free cysteine at RT to allow the formation of GARP-LAP interactions. Then the samples were incubated for 4 hours at 4°C together with magnetic Ni-NTA beads to bind GARP_TS and putative GARP-LAP complexes at the beads. Proteins bound to the beads were analyzed by western blot with anti-Strep-tag antibodies, which could be used to detect both GARP_TS and recombinant TGFβ. (B) Equally treated controls were analyzed on a 10% PAA SDS-PAGE under non-reducing conditions but without the pull-down procedure. After gel electrophoresis, proteins were blotted onto a PVDF membrane and probed with anti-Strep-tag (left) or anti-His-tag antibodies (right), respectively.</p