ALBERT

Description: Introduction: Adenovirus (AdV) infection is a severe complication after hematopoietic stem cell transplantation, particularly in paediatric patients. Control of AdV infection particularly seems to be dependent on CD4+ T-cells but their main AdV antigenic targets have remained unknown so far. In order to design protocols for targeted adoptive immunotherapy the identification of the main AdV antigenic targets is a fundamental prerequisite. Methods: We here adapted a novel technique to directly assess the entire repertoire of CD4+ T-cells specific AdV antigens according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. The cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells were assessed by intracellular analysis of AdV-induced CD154+ CD4+ T-cells. Results: AdV-lysate-specific CD4+ T-cells reacted predominantly with AdV-hexon capsid protein. Furthermore, AdV-hexon (serogroup B) specific CD4+ T-cells crossreacted with recombinant hexon protein derived from various other AdV-serotypes and were characterized by a Th1-like cytokine profile. Conclusion: Our results prove the effectiveness of antigen-induced CD154-expression for assessment of the entire repertoire of CD4+ T-cells specific for pathogens, for the identification of immunodominant target antigen from pathogens. We demonstrate that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive CD4+ T-cells with a Th1-like cytokine profile for adoptive T-cell therapies.

Description: Incidence of febrile neutropenia following cytotoxic treatment of tumor patients can be reduced by prophylactic G-CSF treatment. We report results of a prospective open non-interventional multicenter observational trial in 2233 patients on lenograstim, a recombinant glycoprotein (rHuG-CSF) expressed and glycosylated in Chinese hamster ovary cells. In total 10 846 courses of chemotherapy followed by G-CSF treatment have been documented for either hematologic or oncologic malignancies (breast and ovarian cancer 4207 courses, Hodgkin’s disease and Non Hodgkin’s lymphoma 4381 courses, lung cancer 780 courses, other malignancies 1478 courses, respectively). Lenogratim was administered either interventionally or to less amount for prophylaxis of granulocytopenia; the mean daily dosage was 263 micrograms subcutaneously. A significant number of patients were treated for stage III or IV disease. Almost 1/3 of the patients (34,2%) were elderly aged at least 65 years old. The patients had not been hospitalized for cytotoxic therapy. Lenograstim treatment was started on day 6 (median 6,8 + 4,0) of chemotherapy regimens for almost 5 days (median duration of treatment 5,2 + 3,1 days). Median leukocyte count was 1,9 × 109/L when lenograstim treatment was started, and 5,9 × 109/L at the end of lenograstim treatment, respectively. Especially supporting the CHOP regimen in lymphoma patients lenograstim was administered prophylactically: in 39,5% of lymphoma patients, but only in 24,1% of patients with gynecologic tumors lenograstim treatment was performed for at least 5 days. The rate of infection or fever reported was 9,8% (7,8% in breast cancer patients, 9,0% in lymphoma patients, respectively). 87,1% of chemotherapy courses (87,5% in lymphoma patients and 86,6% in breast cancer patients) could be performed according to the protocol initially scheduled without reduction of dosage or delay of treatment intervals. We conclude that lenograstim was feasible and efficacious for prophylaxis of neutropenic fever in chemotherapy patients. In 2/3 of the patients daily lenograstim dosage of 263 micrograms was suitable to stimulate granulocytopoiesis after conventional cytotoxic therapy within 5 days. No serious adverse events possibly related to lenograstim were reported.

Description: A stepwise approach which combined genome wide expression profiling and a TaqMan realtime PCR based screening was used to identify new markers for the monitoring of minimal residual disease (MRD) in acute myeloid leukemia (AML). Leukemic cells from 52 children with AML were analyzed. Seven genes were identified which are vastly over-expressed in many patients with AML compared to healthy bone marrow: CCL23, GAGED2, MSLN, SPAG6, and ST18 as well as the previously described markers WT1 and PRAME. This set of genes was analyzed in 141 follow-up samples from 25 patients. The expression of all genes decreased to normal levels in patients who achieved a continuous complete remission. Elevated levels of MRD markers were found prior to relapse in 7 out of 10 patients who relapsed. This set of genes should allow a sensitive and specific monitoring of MRD in AML. Notably, some of these markers could also serve as therapeutic targets or might be involved in leukemogenesis. MSLN is already used as a target for immunotherapy in clinical trials in other malignancies. GAGED2 and SPAG6 belong to the family of cancer testis genes which are also studied intensively as targets for immunotherapy. ST18 is a recently discovered tumor suppressor which was not yet described in hematological malignancies. CCL23 is a chemokine that inhibits the proliferation of healthy hematological stem cells. Names, symbols, and geneID of seven MRD markers Gene Symbol Gene Name GeneID CCL23 chemokine (C–C motif) ligand 23 6368 GAGED2 G antigen, family D, 2 9503 MSLN Mesothelin 10232 SPAG6 sperm associated antigen 6 9576 ST18 suppression of tumorigenicity 18 9705 WT1 Wilms tumor 1 7490 PRAME preferentially expressed antigen in melanoma 23532

Description: Introduction: Increasing evidence has emerged that adenoviruses (AdV) can cause severe complications in immunocompromised hosts especially following hematopoietic stem cell transplantation (HSCT). So far protective immune responses in adenovirus infection have not been assessed in detail, but control of infection seems to be dependent on CD4+ T-cells. However, the main antigenic adenovirus targets for specific CD4+ T-cells are unknown. Methods: We here use a novel technique to directly assess the entire repertoire of CD4+ T-cells specific for a defined antigen according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. Additionally, we evaluated the cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells. Results: AdV-lysate-specific CD4+ T-cells reacted efficiently with AdV-hexon and penton proteins. Furthermore, CD4+ T-cells specific for hexon protein derived from AdV-serogroup B crossreact with recombinant hexon protein derived from various other AdV-serotypes and are characterized by a Th1-like cytokine profile. Conclusion: Our results imply that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive proinflammatory CD4+ T-cells for adoptive T-cell therapies.

Description: The IL23R gene on chromosome 1p31 encodes a subunit of the receptor for the proinflammatory cytokine interleukin-23. Recently, IL23R could be identified as a novel inflammatory bowel disease susceptibility gene. The exchange of arginine with glutamine at position 381 of IL23R causes a blockade of the IL-23 signaling pathway which is associated with a reduced risk of both Crohn’s disease and ulcerative colitis. IL-23 is a pivotal cytokine in the differentiation of T cells into inflammatory, IL-17-producing T cells. Because of the requirement for IL-23 in autoimmune disease we hypothesized that IL23R Arg381Gln reduces the risk of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). 141 donors and 141 children (median age, 12 years) with acute lymphoblastic leukemia (n=63), acute myeloid leukemia (n=40), myelodysplastic syndrome (n=26) or chronic myeloid leukemia (n=12) who underwent allogeneic bone marrow (n=93) or peripheral blood stem cell transplantation (n=48; T-cell depleted: n=22) in a single center were genotyped of IL23R for rs11209026 (c.1142G〉A, p.Arg381Gln) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 60% of transplants and HLA-identical related in 30% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 65% of transplants and cyclosporine A alone in 24% of transplants. The IL23R Arg381Gln variant was present in 16 of the 141 donors (11.3%) and in 15 of the 141 patients (10.6%). Interestingly, we found a significantly reduced incidence of acute GVHD grade II–IV in patients who were transplanted from a donor with this specific variant (6.2% versus 33.6%; p=0.025). Furthermore, there was no severe acute GVHD grade III–IV if the variant occurred in the donor (0% versus 14.4%). In three of the donor-patient pairs the variant was present in both individuals and no acute GVHD was observed. The occurrence of the IL23R variant, in either donors or recipients, had no significant impact on chronic GVHD, relapse rate, treatment related mortality, and overall survival. In conclusion, IL23R Arg381Gln variant in the donor confers strong protection against acute GVHD after HSCT in children with hematological malignancies. Therefore, the IL23 signaling pathway seems to play an important role in the pathogenesis of acute GVHD. Blockade of the IL23 receptor by a monoclonal antibody could be a rational therapeutic strategy for acute GVHD.

Description: Abstract 1810 Poster Board I-836 The serum free light chain (SFLC) assay is often though to be a replacement for the cumbersome process of 24-hour urine collection for monoclonal protein estimation (http://www.freelite.co.uk/initialinvestigationpro-24.asp; accessed 16 July 2009) despite evidence suggesting that SFLC estimation cannot replace urine protein quantification (Singhal et al. Blood 2007; 109:3611-2). We studied results from patients with IgG or IgA myeloma if all the following criteria were satisfied: concomitant SFLC and 24-hour urine specimens analyzed, no oligoclonal proteins, no diminished free light chain levels (involved or uninvolved). The aim was to study the correlation between SFLC and 24-hour urine total protein (24UTP), 24-hour urine monoclonal protein (24UMP) and urine immunofixation electrophoresis (UIFE). Only concordant abnormal SFLC ratios were considered abnormal (Singhal et al. Blood 2009; 114:38-9). 558 samples in 114 patients were identified. SFLC ratio was abnormal in 242 and normal in 316 (including 2 discordant abnormal). UIFE (available in 540) was negative in 290. The proportion of samples with normal SFLC ratio decreased as 24UTP increased (P1000 mg - 6% of 32. Similarly, the proportion of samples with normal SFLC ratio declined as 24UMP increased (P=0.001): ≤200 mg (including negative) - 41% of 145, 200-500 mg 4% of 28, 501-1000 mg - 0% of 14, and 〉1000 mg - 0% of 15. SFLC ratio was normal in 84% of samples with negative UIFE and in 26% of samples with positive UIFE (P