TY  - THES
A1  - Munawar, Umair
T1  - Functional analysis of oncogenic lesions in multiple myeloma with potential significance for refractory disease
T1  - Funktionelle Analyse onkogener Läsionen beim Multiplen Myelom mit potenzieller Bedeutung für einen refraktären Krankheitsverlauf
N2  - Despite the advancement in the treatment from genotoxic drugs to more targeted therapies, multiple myeloma (MM) remains incurable. MM is known for its complex genetic heterogeneity as different genetic lesion accrue over the course of the disease. The current work focuses on the functional analysis of genetic lesions found at the time of diagnosis and relapse and their potential role regarding therapy response and refractory disease. Genetic lesions involving tumor suppressor gene TP53, are found at diagnosis and tend to accrue during disease progression. Different types of mono- and biallelic TP53 alterations were emulated in the AMO1 cell line model, were functionally characterized and tested for their potential role in therapy response. Both types of single hit TP53 alteration (deletion 17p and TP53 point mutations) were found to have similar adverse effects on the functionality of the p53 system and response to genotoxic drugs which were completely abolished in the case of double hit TP53 alterations (no p53 expression, or mutant overexpression in wild type TP53 deletion background). Whereas, sensitivity to proteasome inhibitors remained unaltered. Using the clonal competition assay (CCA), single TP53 hit clones were found to have a fitness advantage over wildtype cells. Proliferative cell fitness was further enhanced in double hit TP53 clones, as they dominated wildtype and single hit TP53 clones in the CCA. Presence of external selection pressure in the form of low dose melphalan expedited the intrinsic fitness advantage. Alterations found in CUL4B, a component of CRL4-CRBN protein complex, a target of immunomodulatory drugs (IMiDs), were also functionally analyzed in the current study. Hotspot mutations and mutations found in IMiDs refractory patients were modelized in L363 cells and their role in IMiDs sensitivity was studied. CUL4B mutations were found not to be involved in providing lenalidomide resistance to the cell, whereas knocking CUL4B out was observed to provide negative fitness to the cells in CCA. In the presence of external selection pressure, these clones showed fitness, which was lost in the case of lenalidomide withdrawal. This shows that some alterations may play a role in refractory patients only in the presence of therapy, and as soon as therapy is discontinued, these altered clones may disappear such as clones with alterations in CUL4B. On the other hand, some alterations provide drug-independent intrinsic positive fitness, however, be further enhanced by drug exposure, such as seen in case of TP53 altered clones. Therefore, close monitoring and functional analysis of evolving clones is desired during disease progression, as it can be helpful in therapeutic guidance to achieve a better outcome for patients.
N2  - Das Multiple Myelom (MM) ist im Normalfall eine unheilbare Erkrankung, trotz etlicher Fortschritte hinsichtlich der Behandlung, angefangen von genotoxischen Medikamenten bis hin zu zielgerichteten Therapien. Eine komplexe genetische Heterogenität, bei der sich Läsionen im Verlauf der Krankheit ansammeln, ist typisch für das MM. Diese Arbeit beschäftigt sich mit der funktionellen Analyse von genetischen Läsionen zum Erstdiagnose-Zeitpunkt bzw. Rezidiv und ihrer potenziellen Bedeutung bezüglich des Ansprechens auf die Therapie und einer refraktären Verlaufsform. Genetische Veränderungen des Tumorsuppressorgens TP53 sind mitunter schon bei der Erst-Diagnose nachweisbar und nehmen im Krankheitsverlauf weiter zu. Deshalb wurden verschiedene mono- und biallelische TP53 Mutationen in AMO1 Zelllinien-Modellen nachgebildet, funktionell charakterisiert und auf ihre potenzielle Rolle im Therapie-Ansprechen getestet. Dabei wurden für beide Formen von single hit TP53 Alterationen (17p Deletionen und TP53 Punktmutationen) ähnlich nachteilige Effekte auf die Funktionalität des p53 Systems und das Ansprechen auf genotoxische Medikation gefunden. Im Falle von double hit TP53 Alterationen (keinerlei p53 Expression oder aber hohe Spiegel von mutiertem p53 bei TP53 wildtype deletierem Hintergrund) wurden diese Effekte noch weiter verstärkt. Die Sensitivität gegenüber Proteasom-Inhibitoren blieb indessen unbeeinträchtigt. Mithilfe des Clonal Competition Assays (CCA), wurde festgestellt, dass single hit TP53 Klone gegenüber wildtypischen Zellen einen Fitnessvorteil haben. In double hit TP53 Klonen war die proliferative Zell-Fitness zu dem gesteigert, sodass diese über wildtypische und single hit TP53 Klone im CCA dominierten. Die Anwesenheit eines externen Selektionsdrucks in Form von Niedrigdosis-Melphalan verstärkte zusätzlich den intrinsischen Fitnessvorteil. Aberrationen in CUL4B, einer Untereinheit des CRL4-CRBN Protein Komplexes, dem Angriffspunkt der Immunmodulatoren (IMiDs), wurden in dieser Studie ebenfalls funktionell analysiert. Hotspot Mutationen und solche Mutationen, die gehäuft in IMiD refraktären Patienten auftreten, wurden in L363 Zellen modelliert und hinsichtlich ihres Einflusses auf die IMiD Sensitivität untersucht. CUL4B Mutationen waren nicht in Lenalidomid-Resistenz involviert, ein Knockout von CUL4B äußerte sich aber in einer negativen Allgemeinzustand der Zellen im CCA. In Anwesenheit eines externen Selektionsdrucks, zeigten diese Klone einen Fitnessvorteil, der wieder verloren ging, wenn kein Lenalidomid mehr zugegeben wurde. Dies zeigt, dass manche Alterationen nur dann eine Rolle in refraktären Patienten zu spielen scheinen, wenn diese therapiert werden. Sobald die Therapie eingestellt oder unterbrochen wird, könnten solche Klone verschwinden wie z.B. CUL4B-Alterationen. Auf der anderen Seite bedingen manche Veränderungen wiederum einen Medikamenten-unabhängigen, intrinsisch Fitnessvorteil, der jedoch durch Medikamenten-Exposition noch zusätzlich verstärkt werden kann, wie beispielweise bei TP53 mutierten Klonen. Daher ist eine engmaschige Überwachung während der Krankheits-Progression sowie eine funktionelle Analyse der sich entwickelnden Klone wünschenswert. Dies könnte für die therapeutische Beratung hilfreich sein um somit ein besseres Behandlungsergebnis für die Patienten zu erzielen.
KW  - TP53
KW  - Myeloma
KW  - Cul4b
KW  - MM
KW  - TP53 lesions
KW  - Clonal competition assay
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216446
ER  - 
TY  - JOUR
A1  - Steinhardt, M. J.
A1  - Wiercinska, E.
A1  - Pham, M.
A1  - Grigoleit, G. U.
A1  - Mazzoni, A.
A1  - Da-Via, M.
A1  - Zhou, X.
A1  - Meckel, K.
A1  - Nickel, K.
A1  - Duell, J.
A1  - Krummenast, F. C.
A1  - Kraus, S.
A1  - Hopkinson, C.
A1  - Weissbrich, B.
A1  - Müllges, W.
A1  - Stoll, G.
A1  - Kortüm, K. M.
A1  - Einsele, H.
A1  - Bonig, H.
A1  - Rasche, L.
T1  - Progressive multifocal leukoencephalopathy in a patient post allo-HCT successfully treated with JC virus specific donor lymphocytes
JF  - Journal of Translational Medicine
N2  - Background 
Progressive multifocal leukoencephalopathy is a demyelinating CNS disorder. Reactivation of John Cunningham virus leads to oligodendrocyte infection with lysis and consequent axonal loss due to demyelination. Patients usually present with confusion and seizures. Late diagnosis and lack of adequate therapy options persistently result in permanent impairment of brain functions. Due to profound T cell depletion, impairment of T-cell function and potent immunosuppressive factors, allogeneic hematopoietic cell transplantation recipients are at high risk for JCV reactivation. To date, PML is almost universally fatal when occurring after allo-HCT. 

Methods 
To optimize therapy specificity, we enriched JCV specific T-cells out of the donor T-cell repertoire from the HLA-identical, anti-JCV-antibody positive family stem cell donor by unstimulated peripheral apheresis [1]. For this, we selected T cells responsive to five JCV peptide libraries via the Cytokine Capture System technology. It enables the enrichment of JCV specific T cells via identification of stimulus-induced interferon gamma secretion. 

Results 
Despite low frequencies of responsive T cells, we succeeded in generating a product containing 20 000 JCV reactive T cells ready for patient infusion. The adoptive cell transfer was performed without complication. Consequently, the clinical course stabilized and the patient slowly went into remission of PML with JCV negative CSF and containment of PML lesion expansion. 

Conclusion 
We report for the first time feasibility of generating T cells with possible anti-JCV activity from a seropositive family donor, a variation of virus specific T-cell therapies suitable for the post allo transplant setting. We also present the unusual case for successful treatment of PML after allo-HCT via virus specific T-cell therapy.
KW  - Myeloma
KW  - JCV
KW  - Prodigy
KW  - CCS
KW  - PML
KW  - Donor lymphocytes
KW  - Adaptive cell transfer
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229307
VL  - 18
ER  - 
TY  - JOUR
A1  - Mitchell, Jonathan S.
A1  - Li, Ni
A1  - Weinhold, Niels
A1  - Försti, Asta
A1  - Ali, Mina
A1  - van Duin, Mark
A1  - Thorleifsson, Gudmar
A1  - Johnson, David C.
A1  - Chen, Bowang
A1  - Halvarsson, Britt-Marie
A1  - Gudbjartsson, Daniel F.
A1  - Kuiper, Rowan
A1  - Stephens, Owen W.
A1  - Bertsch, Uta
A1  - Broderick, Peter
A1  - Campo, Chiara
A1  - Einsele, Hermann
A1  - Gregory, Walter A.
A1  - Gullberg, Urban
A1  - Henrion, Marc
A1  - Hillengass, Jens
A1  - Hoffmann, Per
A1  - Jackson, Graham H.
A1  - Johnsson, Ellinor
A1  - Jöud, Magnus
A1  - Kristinsson, Sigurdur Y.
A1  - Lenhoff, Stig
A1  - Lenive, Oleg
A1  - Mellqvist, Ulf-Henrik
A1  - Migliorini, Gabriele
A1  - Nahi, Hareth
A1  - Nelander, Sven
A1  - Nickel, Jolanta
A1  - Nöthen, Markus M.
A1  - Rafnar, Thorunn
A1  - Ross, Fiona M.
A1  - da Silva Filho, Miguel Inacio
A1  - Swaminathan, Bhairavi
A1  - Thomsen, Hauke
A1  - Turesson, Ingemar
A1  - Vangsted, Annette
A1  - Vogel, Ulla
A1  - Waage, Anders
A1  - Walker, Brian A.
A1  - Wihlborg, Anna-Karin
A1  - Broyl, Annemiek
A1  - Davies, Faith E.
A1  - Thorsteinsdottir, Unnur
A1  - Langer, Christian
A1  - Hansson, Markus
A1  - Kaiser, Martin
A1  - Sonneveld, Pieter
A1  - Stefansson, Kari
A1  - Morgan, Gareth J.
A1  - Goldschmidt, Hartmut
A1  - Hemminki, Kari
A1  - Nilsson, Björn
A1  - Houlston, Richard S.
T1  - Genome-wide association study identifies multiple susceptibility loci for multiple myeloma
JF  - Nature Communications
N2  - Multiple myeloma (MM) is a plasma cell malignancy with a significant heritable basis. Genome-wide association studies have transformed our understanding of MM predisposition, but individual studies have had limited power to discover risk loci. Here we perform a meta-analysis of these GWAS, add a new GWAS and perform replication analyses resulting in 9,866 cases and 239,188 controls. We confirm all nine known risk loci and discover eight new loci at 6p22.3 (rs34229995, P=1.31 × 10−8), 6q21 (rs9372120, P=9.09 × 10−15), 7q36.1 (rs7781265, P=9.71 × 10−9), 8q24.21 (rs1948915, P=4.20 × 10−11), 9p21.3 (rs2811710, P=1.72 × 10−13), 10p12.1 (rs2790457, P=1.77 × 10−8), 16q23.1 (rs7193541, P=5.00 × 10−12) and 20q13.13 (rs6066835, P=1.36 × 10−13), which localize in or near to JARID2, ATG5, SMARCD3, CCAT1, CDKN2A, WAC, RFWD3 and PREX1. These findings provide additional support for a polygenic model of MM and insight into the biological basis of tumour development.
KW  - Cancer genetics
KW  - Genome-wide association studies
KW  - Myeloma
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165983
VL  - 7
ER  -