PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

Analysis of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-reactive CD8(+) T cell responses in children with NPM-ALK(+) anaplastic large cell lymphoma.

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

Effective childhood cancer treatment: the impact of large scale clinical trials in Germany and Austria.

In Germany and Austria, more than 90% of pediatric cancer patients are enrolled into nationwide disease-specific first-line clinical trials or interim registries. Essential components are a pediatric cancer registry and centralized reference laboratories, imaging review, and tumor board assistance. The five-year overall survival rate in countries where such infrastructures are established has improved from <20% before 1950 to >80% since 1995. Today, treatment intensity is tailored to the individual patient's risk to provide the highest chances of survival while minimizing deleterious late effects. Multicenter clinical trials are internationalized and serve as platforms for further improvements by novel drugs and biologicals.

Use of minimal disseminated disease and immunity to NPM-ALK antigen to stratify ALK-positive ALCL patients with different prognosis.

We studied the prognostic value of minimal disseminated disease (MDD) and anti-ALK immune response in children with NPM-ALK-positive anaplastic-large cell lymphoma (ALCL) and evaluated their potential for risk stratification. NPM-ALK transcripts were analyzed by RT-PCR in bone marrow/peripheral blood of 128 ALCL patients at diagnosis, whereas ALK antibody titers in plasma were assessed using an immunocytochemical approach. MDD was positive in 59% of patients and 96% showed an anti-ALK response. Using MDD and antibody titer results, patients could be divided into three biological risk groups (bRG) with different prognosis: high risk (bHR): MDD-positive and antibody titer ≤ 1/750, 26/128 (20%); low risk (bLR): MDD negative and antibody titer >1/750, 40/128 (31%); intermediate risk (bIR): all remaining patients, 62/128 (48%). Progression-free survival was 28% (s.e., 9%), 68% (s.e., 6%) and 93% (s.e., 4%) for bHR, bIR and bLR, respectively (P<0.0001). Survival was 71% (s.e., 9%), 83% (s.e., 5%) and 98% (s.e., 2%) for bHR, bIR and bLR (P=0.02). Only bHR and histology other than common type were predictive of higher risk of failure (hazard ratio 4.9 and 2.7, respectively) in multivariate analysis. Stratification of ALCL patients based on MDD and anti-ALK titer should be considered in future ALCL trials to optimize treatment.

Human leukocyte antigen distribution in German Caucasians with advanced Ewing's sarcoma.

BACKGROUND: Risk stratification criteria for patients with Ewing's sarcoma family of tumors (ESFT) are still limited. We hypothesized divergent human leukocyte antigen (HLA) patterns in ESFT patients and compared HLA-A, -B and -DR phenotype frequencies of patients with advanced ESFT with those of healthy controls. PATIENTS: HLA types of all German Caucasian patients with advanced ESFT and available HLA-A, -B and -DR data registered in the European Group for Blood and Marrow Transplantation, Paediatric Registry for Stem Cell Transplantation and the MetaEICESS data bases (study group, n=30) were retrospectively compared with HLA types of healthy German stem cell donors (control group, n=8 862 for single HLA frequencies and n=8 839 for allele combinations). Study group patients had been immuno-typed due to eligibility for allogeneic stem cell transplantation for high risk of treatment failure, and thus constituted a selected subgroup of ESFT patients. RESULTS: After Bonferroni correction for multiple testing (PC), phenotype frequencies of HLA-A24 remained significantly higher in the study group compared to controls (PC<0.05). Furthermore, several HLA combinations were significantly more frequent in the study group compared to controls (all PC<0.05). CONCLUSION: We report an increased incidence of circumscribed HLA patterns in German Caucasians with advanced ESFT. The possible clinical significance of this observation has to be re-assessed in prospective trials comprising larger ESFT patient numbers of all risk groups.

Non-Hodgkin's lymphoma in adolescents: experiences in 378 adolescent NHL patients treated according to pediatric NHL-BFM protocols.

Age-related differences in the distribution, biology and treatment response of non-Hodgkin's lymphoma (NHL) in adolescents remain to be elucidated. The current analyses present clinical parameters and outcomes of adolescents treated in pediatric NHL-BFM trials. Patients were stratified by histological subtype: lymphoblastic lymphoma (LBL); mature B-NHL, including Burkitt's lymphoma/leukemia (BL/B-AL), diffuse B-cell lymphoma (DLBCL-CB) and mediastinal B-cell lymphoma (PMLBL); and anaplastic large cell lymphoma (ALCL). Between October 1986 and December 2007, 2915 patients were registered, including 378 (13%) adolescents (15-18 years) with BL/B-AL (n=101), ALCL (n=74), DLBCL-CB (n=55), T-LBL (n=45), PMLBL (n=24), pB-LBL (n=13) and rare or not-specified NHL subtypes (n=66). The 5-year event-free survival (EFS) was 79±2% for adolescents compared with 85±1% for patients aged <15 years (P=0.014). EFS was 83±7% for adolescents with T-LBL, 82±4% with BL/B-AL, 85±5% with DLBCL-CB, 57±10% with PMLBL and 70±6% with ALCL. According to sex, the 5-year EFS in females versus males, respectively, was 70±5 versus 83±2% overall (P=0.004), 57±17 versus 92±6% (P=0.0036) for T-LBL patients and 71±9 versus 97±3% (P=0.0067) for DLBCL-CB patients. Adolescents with NHL treated according to pediatric NHL-BFM protocols had an EFS of 79±2%, which is marginally inferior to that of children. In adolescents with T-LBL and DLBCL-CB, female sex was associated with a worse prognosis.

Improved outcome of treatment-resistant high-risk Langerhans cell histiocytosis after allogeneic stem cell transplantation with reduced-intensity conditioning.

Children with multisystem Langerhans cell histiocytosis (LCH) and risk organ involvement who fail to respond to conventional chemotherapy have an extremely poor prognosis. Myeloablative stem cell transplantation (SCT) as a possible salvage approach for these patients has been associated with a high risk of transplant-related mortality. Therefore, allogeneic stem cell transplantation following a reduced-intensity conditioning regimen (RIC-SCT) has recently been performed as an alternative salvage approach. We report on the experience with allogeneic RIC-SCT in nine pediatric high-risk LCH patients. Conditioning regimen included fludarabine in all patients, melphalan in eight patients, total lymphoid irradiation in six patients, total body irradiation in two, antithymocyte globulin in five, and Campath in four patients. RIC-SCT was well tolerated with regard to common procedure-related complications. Two patients died 50 and 69 days after RIC-SCT, respectively. Seven out of the nine patients survived and showed no signs of disease activity (including one with nonengraftment and full autologous hematopoietic recovery) after median follow-up of 390 days post-SCT. Based on this observation, we conclude that RIC-SCT is a feasible procedure with low transplant-related morbidity and mortality and a promising new salvage approach for high-risk LCH patients with resistant risk organ involvement.

Inactivation of the ARF-MDM-2-p53 pathway in sporadic Burkitt's lymphoma in children.

Burkitt's lymphomas (BLs) are characterized by an activated MYC gene that provides a constitutive proliferative signal. However, activated myc can initiate ARF-dependent activation of p53 and apoptosis as well. Data derived from cell culture and animal models suggest that the inactivation of the ARF-MDM-2-p53 apoptotic signaling pathway may be a necessary secondary event for the development of BL. This has not been tested in freshly excised BL tissue. We investigated the ARF-MDM-2-p53 pathway in tumor specimen from 24 children with sporadic BL/B-ALL. Direct sequencing revealed a point mutation in the p53 gene in four BL. Overexpression of MDM-2 was evident in 10 of the BL samples analyzed by real-time quantitative PCR. Deletion of the CDKN2A locus that encodes ARF or reduced expression of ARF could not be detected in any BL by fluorescence in situ hybridization analysis or real-time quantitative PCR, respectively. Our results indicate that the ARF-MDM-2-p53 apoptotic pathway is disrupted in about 55% of the cases of childhood sporadic BL. We suggest that in addition to the inactivation of the ARF-MDM-2-p53 protective checkpoint function other antiapoptotic mutations may occur in a substantial part of children with sporadic BL.

Design and evaluation of chemically synthesized siRNA targeting the NPM-ALK fusion site in anaplastic large cell lymphoma (ALCL).

The NPM-ALK fusion protein is found in up to 75% of pediatric anaplastic large cell lymphomas (ALCL). The ALK kinase becomes constitutively activated and triggers malignant transformation. Molecular targeting of the tumor-specific NPM-ALK fusion by gene-silencing methods seems to be a promising approach both for the treatment of ALCL and to decipher signaling pathways used by NPM-ALK. We designed and evaluated three chemically synthesized small interfering RNAs (siRNAs) for downregulation of the NPM-ALK fusion mRNA. Compared to HeLa cells transfected with the NPM-ALK expression plasmid only and to an siRNA containing two point mutations, the most potent anti-NPM-ALK siRNA reduced NPM-ALK protein expression in HeLa cells to almost undetectable levels, and the number of cells stained positively for NPM-ALK decreased by 80%. With respect to signaling, expressing of NPM-ALK increased the activity of AKT and ERK in HeLa cells, and this effect could be blocked by the specific siRNA targeting NPM-ALK. Expression of endogenous NPM-ALK mRNA in SR786 ALCL cells decreased by 50%-60% in cells transfected with the NPM-ALK siRNA. However, the amount of NPM-ALK protein was not influenced by a single transfection of the siRNAs against NPM-ALK. Repeated transfections over 8 days led to a significant reduction in NPM-ALK protein but without induction of apoptosis. We believe that the long protein half-life of NPM-ALK, at least 48 hours, limits the application of transiently transfected siRNAs. Nevertheless, RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against NPM-ALK-positive lymphomas.

Role of ERK activation in growth and erythroid differentiation of K562 cells.

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24-48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.

An essential role for mitogen-activated protein kinases, ERKs, in preventing heat-induced cell death.

Stimulation of mitogen-activated protein kinases (MAPKs) or extracellular signal regulated protein kinases (ERKs) after exposure of mammalian cells to ultraviolet (UV) and X-irradiation occurs through activation of receptor tyrosine kinases via Ras/Raf/Mek/ERKs cascade. This activation of MAPKs is proposed to play a role in the replacement of damaged proteins during these stresses. Heat shock also activates MAPKs; however, the signaling cascade and the biochemical and physiological links between activation by heat and downstream effects are unknown. In this report we demonstrate that, unlike irradiation, heat induces MAPKs through ceramide metabolism to sphingosine with stimulation of Raf-1 protein kinase. The activation of MAPKs by heat does not occur in all cell types, because the step(s) downstream of ceramide to activation of Raf-1 protein kinase is missing in myeloid leukemic cells such as HL-60, U937, and K562, while it is present in NIH3T3 fibroblasts. Heat-induced MAPK activation may enhance the ability of cells to survive a severe heat shock. Blocking 60-70% of the activity of MAPK (ERK1) by stable overexpression of the dominant negative allele ERK1-KR renders NIH3T3 and K562 cells up to 100-fold more sensitive to cytotoxic effects of heat. Conversely, NIH3T3 and K562 cells stably overexpressing the wild-type ERK1 develop resistance to killing by heat. These results suggest that increased thermal sensitivity of leukemic cells to thermal stress or other cancer therapy regimens could be attributable to lack of pertinent activation of the MAPK pathway by such stresses.