Elke Rücker-Braun

TP53 mutation in patients with high-risk acute myeloid leukaemia treated with allogeneic haematopoietic stem cell transplantation

Treatment success in patients with acute myeloid leukaemia (AML) is heterogeneous. Cytogenetic and molecular alterations are strong prognostic factors, which have been used to individualize treatment. Here, we studied the impact of TP53 mutations on the outcome of AML patients with adverse cytogenetic risk treated with allogeneic haematopoietic stem cell transplantation (HSCT). Samples of 97 patients with AML and adverse‐risk cytogenetics who had received a HSCT within three randomized trials were analysed. Complete sequencing of the TP53 coding region was performed using next generation sequencing. The median age was 51 years. Overall, TP53 mutations were found in 40 patients (41%). With a median follow up of 67 months, the three‐year probabilities of overall survival (OS) and event‐free survival for patients with TP53 wild type were 33% [95% confidence interval (CI), 21% to 45%] and 24% (95% CI, 13% to 35%) compared to 10% (95% CI, 0% to 19%) and 8% (95% CI, 0% to 16%) (P = 0·002 and P = 0·007) for those with mutated TP53, respectively. In multivariate analysis, the TP53‐mutation status had a negative impact on OS (Hazard Ratio = 1·7; P = 0·066). Mutational analysis of TP53 might be an important additional tool to predict outcome after HSCT in patients with adverse karyotype AML.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

Allogeneic stem cell transplantation is potentially curative, but associated with post‐transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR‐α repertoire and its clinical relevance in patients following stem cell transplantation. Using next‐generation sequencing we examined the TCR‐α repertoire of CD8+ T cells and CMV‐specific CD8+ T cells in four patients. Additionally, we performed single‐cell TCR‐αβ sequencing of CMV‐specific CD8+ T cells. The TCR‐α composition of human leucocyte antigen (HLA)‐A*0201 CMVpp65– and CMVIE‐specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8+ T cells and half of the total CD8+ T cell repertoire was dominated by few CMV‐reactive clonotypes. Some TCR‐α clonotypes were shared between patients. Gene expression of the circulating CMV‐specific CD8+ T cells was consistent with chronically activated effector memory T cells. The CD8+ T cell response to CMV reactivation resulted in an expansion of a few TCR‐α clonotypes to dominate the CD8+ repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti‐viral T cell response in this setting.

Abundant CMV reactive clonotypes in the CD8+ T cell receptor alpha repertoire following allogeneic transplantation

Allogeneic stem cell transplantation is potentially curative, but associated with post‐transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR‐α repertoire and its clinical relevance in patients following stem cell transplantation. Using next‐generation sequencing we examined the TCR‐α repertoire of CD8+ T cells and CMV‐specific CD8+ T cells in four patients. Additionally, we performed single‐cell TCR‐αβ sequencing of CMV‐specific CD8+ T cells. The TCR‐α composition of human leucocyte antigen (HLA)‐A*0201 CMVpp65– and CMVIE‐specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8+ T cells and half of the total CD8+ T cell repertoire was dominated by few CMV‐reactive clonotypes. Some TCR‐α clonotypes were shared between patients. Gene expression of the circulating CMV‐specific CD8+ T cells was consistent with chronically activated effector memory T cells. The CD8+ T cell response to CMV reactivation resulted in an expansion of a few TCR‐α clonotypes to dominate the CD8+ repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti‐viral T cell response in this setting.

Generation of high-avidity, WT1-reactive CD8+ cytotoxic T cell clones with anti-leukemic activity by streptamer technology

Antje Tunger, Rebekka Wehner, Malte von Bonin, Denise K€ uhn, Falk Heidenreich, Sarah Matko, Magdalena Nauerth, Elke R€ ucker-Braun, Sevina Dietz, Cornelia S. Link, Anne Eugster, Marcus Odendahl, Dirk H. Busch, Torsten Tonn, Ezio Bonifacio, Lothar Germeroth, Johannes Schetelig, Michael P. Bachmann, Martin Bornh€auser and Marc Schmitz Institute of Immunology, Medical Faculty, TU Dresden, Dresden, Germany; National Center for Tumor Diseases, University Hospital Carl Gustav Carus, TU Dresden, Germany; Department of Medicine I, University Hospital of Dresden, Dresden, Germany; German Cancer Consortium (DKTK), Dresden, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; Center for Regenerative Therapies Dresden (CRTD), Medical Faculty, TU Dresden, Dresden, Germany; Institute of Transfusion Medicine, German Red Cross Blood Donation Service North-East, Dresden, Germany; Institute for Medical Microbiology, Immunology and Hygiene, TU Munich, Munich, Germany; Juno Therapeutics GmbH, G€ottingen, Germany; Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz Center Dresden-Rossendorf, Dresden, Germany

Pilot Study on Mass Spectrometry–Based Analysis of the Proteome of CD34+CD123+ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia

Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrometry methods to analyze the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy, granulocyte-colony stimulating factor (G-CSF) mobilized stem cell donors were analyzed. In this Tandem Mass Tag (TMT) 10-plex labelling–based approach, 2070 proteins were identified with 171 proteins differentially abundant in one or both cellular compartments. This proof-of-principle-study demonstrates the potential of mass spectrometry to detect differentially expressed proteins in two compartment fractions of the entire proteome of leukemic stem cells, compared to their non-malignant counterparts. This may contribute to future immunotherapeutic target discoveries and individualized AML patient characterization.

T cell receptor alpha repertoire of CD8+ T cells following allogeneic stem cell transplantation using next-generation sequencing

Alloreactivity or opportunistic infections following allogeneic stem cell transplantation are difficult to predict and contribute to post-transplantation mortality. How these immune reactions result in changes to the T-cell receptor repertoire remains largely unknown. Using next-generation sequencing, the T-cell receptor alpha (TRα) repertoire of naïve and memory CD8+ T cells from 25 patients who had received different forms of allogeneic transplantation was analyzed. In parallel, reconstitution of the CD8+/CD4+ T-cell subsets was mapped using flow cytometry. When comparing the influence of anti-T-cell therapy, a delay in the reconstitution of the naïve CD8+ T-cell repertoire was observed in patients who received in vivo T-cell depletion using antithymocyte globulin or post-transplantation cyclophosphamide in case of haploidentical transplantation. Sequencing of the TRα identified a repertoire consisting of more dominant clonotypes (>1% of reads) in these patients at 6 and 18 months post transplantation. When comparing donor and recipient, approximately 50% and approximately 80% of the donors’ memory repertoire were later retrieved in the naïve and memory CD8+ T-cell receptor repertoire of the recipients, respectively. Although there was a remarkable expansion of single clones observed in the recipients’ memory CD8+ TRα repertoire, no clear association between graft-versus-host disease or cytomegalovirus infection and T-cell receptor diversity was identified. A lower TRα diversity was observed in recipients of a cytomegalovirus-seropositive donor (P=0.014). These findings suggest that CD8+ T-cell reconstitution in transplanted patients is influenced by the use of T-cell depletion or immunosuppression and the donor repertoire.

Assessment of the T cell receptor repertoire in long-term platelet donors by next generation sequencing.

Temporary suppression of circulating antiplatelet alloantibodies by the massive infusion of fresh, stored, or lyophilized platelets. Transfusion, 18, 429–435. Narvios, A., Reddy, V., Martinez, F. & Lichtiger, B. (2005) Slow infusion of platelets: a possible alternative in the management of refractory thrombocytopenic patients. American Journal of Hematology, 79, 80. Stanworth, S.J., Navarrete, C., Estcourt, L. & Marsh, J. (2015) Platelet refractoriness–practical approaches and ongoing dilemmas in patient management. British Journal of Haematology, 171, 297–305. Vassallo, R. (2013) Management of the platelettransfusion-refractory patient. In: Platelet Transfusion Therapy (eds. by Sweeney, J.D. & Lozano, M.), pp. 321–358. AABB Press, Bethesda (MD).

Mass spectrometry-based identification of a naturally presented receptor tyrosine kinase-like orphan receptor 1-derived epitope recognized by CD8+ cytotoxic T cells

Despite recent treatment improvements with the approval of cell signaling pathway inhibitors, additional treatment options are needed for patients with relapsed/refractory high-risk chronic lymphocytic leukemia (CLL). An emerging immunotherapeutic strategy for CLL is based on the adoptive transfer