Conversion of AML-blasts to leukemia-derived dendritic cells (DCleu) in 'DC-culture-media' shifts correlations of released chemokines with antileukemic T-cell reactions.

Dendritic cells (DC) and T-cells are mediators of CTL-responses. Autologous (from patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS)) or allogeneic (donor)-T-cells stimulated by DCleu, gain an efficient lysis of naive blasts, although not in every case. CXCL8, -9, -10, CCL2, -5 and Interleukin (IL-12) were quantified by Cytometric Bead Array (CBA) in supernatants from 5 DC-generating methods and correlated with AML-/MDS-patients' serum-values, DC-/T-cell-interactions/antileukemic T-cell-reactions after mixed lymphocyte culture (MLC) and patients' clinical course. The blast-lytic activity of T-cells stimulated with DC or mononuclear cells (MNC) was quantified in a cytotoxicity assay. Despite great variations of chemokine-levels, correlations with post-stimulation (after stimulating T-cells with DC in MLC) improved antileukemic T-cell activity were seen: higher released chemokine-values correlated with improved T-cells' antileukemic activity (compared to stimulation with blast-containing MNC) - whereas with respect to the corresponding serum values higher CXCL8-, -9-, and -10- but lower CCL5- and -2-release correlated with improved antileukemic activity of DC-stimulated (vs. blast-stimulated) T-cells. In DC-culture supernatants higher chemokine-values correlated with post-stimulation improved antileukemic T-cell reactivity, whereas higher serum-values of CXCL8, -9, and -10 but lower serum-values of CCL5 and -2 correlated with post-stimulation improved antileukemic T-cell-reactivity. In a context of 'DC'-stimulation (vs serum) this might point to a change of (CCL5 and -2-associated) functionality from a more 'inflammatory' or 'tumor-promoting' to a more 'antitumor'-reactive functionality. This knowledge could contribute to develop immune-modifying strategies that promote antileukemic (adaptive) immune-responses.

Serum Chemokine-release Profiles in AML-patients Might Contribute to Predict the Clinical Course of the Disease.

In cancer or hematologic disorders, chemokines act as growth- or survival factors, regulating hematopoiesis and angiogenesis, determining metastatic spread and controlling leukocyte infiltration into tumors to inhibit antitumor immune responses. The aim was to quantify the release of CXCL8, -9, -10, CCL2, -5, and IL-12 in AML/MDS-pts' serum by cytometric bead array and to correlate data with clinical subtypes and courses. Minimal differences in serum-levels subdivided into various groups (e.g. age groups, FAB-types, blast-proportions, cytogenetic-risk-groups) were seen, but higher release of CXCL8, -9, -10 and lower release of CCL2 and -5 tendentially correlated with more favorable subtypes (<50 years of age, <80% blasts in PB). Comparing different stages of the disease higher CCL5-release in persisting disease and a significantly higher CCL2-release at relapse were found compared to first diagnosis - pointing to a change of 'disease activity' on a chemokine level. Correlations with later on achieved response to immunotherapy and occurrence of GVHD were seen: Higher values of CXCL8, -9, -10 and CCL2 and lower CCL5-values correlated with achieved response to immunotherapy. Predictive cut-off-values were evaluated separating the groups in 'responders' and 'non-responders'. Higher levels of CCL2 and -5 but lower levels of CXCL8, -9, -10 correlated with occurrence of GVHD. We conclude, that in AML-pts' serum higher values of CXCL8, -9, -10 and lower values of CCL5 and in part of CCL2 correlate with more favorable subtypes and improved antitumor'-reactive function. This knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune responses.

Paraneoplastic limbic encephalitis with SOX1 and PCA2 antibodies and relapsing neurological symptoms in an adolescent with Hodgkin lymphoma.

BACKGROUND: Immune cross-reactivity between malignant and normal tissues causes the rare, so called paraneoplastic syndrome (PS). In approximately 60% of the patients, various onconeural antibodies are detectable in the cerebrospinal fluid (CSF) and are associated with typical tumour entities. METHODS: We report an unusual case of paraneoplastic limbic encephalitis (PLE) in a 17-year-old adolescent with classical Hodgkin lymphoma. RESULTS: He presented with a variety of neurologic and neuropsychiatric symptoms, profound B-symptoms and typical MRI findings including hyperintense lesions with contrast enhancement in the medial temporal lobe and limbic system. Under immunosuppressive therapy and subsequently chemotherapy the neurological situation only temporarily improved and worsened again after interruption of immunosuppression several times. Thus, multiple courses of multidrug immunosuppressive therapy were administered. To date, five years after initial presentation, the young man is able to walk with walking aids and orthoses and is still on oral prednisolone therapy. Analyses of the CSF and serum revealed anti SOX-1 antibodies at initial presentation but PCA-2 antibodies seven months after diagnosis. CONCLUSION: Neurologic and/or neuropsychiatric symptoms combined with typical MRI findings should raise the suspicion of PS and lead to further diagnostics for an underlying tumour even in children.

Control of AC133/CD133 and impact on human hematopoietic progenitor cells through nucleolin.

AC133 is a prominent surface marker of CD34+ and CD34- hematopoietic stem/progenitor cell (HSPC) subsets. AC133+ HSPCs contain high progenitor cell activity and are capable of hematopoietic reconstitution. Furthermore, AC133 is used for prospective isolation of tumor-initiating cells in several hematological malignancies. Nucleolin is a multifunctional factor of growing and cancer cells, which is aberrantly active in certain hematological neoplasms, and serves as a candidate molecular target for cancer therapy. Nucleolin is involved in gene transcription and RNA metabolism and is prevalently expressed in HSPCs, as opposed to differentiated hematopoietic tissue. The present study dissects nucleolin-mediated activation of surface AC133 and its cognate gene CD133, via specific interaction of nucleolin with the tissue-dependent CD133 promoter P1, as a mechanism that crucially contributes to AC133 expression in CD34+ HSPCs. In mobilized peripheral blood (MPB)-derived HSPCs, nucleolin elevates colony-forming unit (CFU) frequencies and enriches granulocyte-macrophage CFUs. Furthermore, nucleolin amplifies long-term culture-initiating cells and also promotes long-term, cytokine-dependent maintenance of hematopoietic progenitor cells. Active ß-catenin, active Akt and Bcl-2 levels in MPB-derived HSPCs are nucleolin-dependent, and effects of nucleolin on these cells partially rely on ß-catenin activity. The study provides new insights into molecular network relevant to stem/progenitor cells in normal and malignant hematopoiesis.

Monitoring minimal residual disease in children with high-risk relapses of acute lymphoblastic leukemia: prognostic relevance of early and late assessment.

The prognosis for children with high-risk relapsed acute lymphoblastic leukemia (ALL) is poor. Here, we assessed the prognostic importance of response during induction and consolidation treatment prior to hematopoietic stem cell transplantation (HSCT) aiming to evaluate the best time to assess minimal residual disease (MRD) for intervention strategies and in future trials in high-risk ALL relapse patients. Included patients (n=125) were treated uniformly according to the ALL-REZ BFM (Berlin-Frankfurt-Münster) 2002 relapse trial (median follow-up time=4.8 years). Patients with MRD ⩾10(-3) after induction treatment (76/119, 64%) or immediately preceding HSCT (19/71, 27%) had a significantly worse probability of disease-free survival 10 years after relapse treatment begin, with 26% (±6%) or 23% (±7%), respectively, compared with 58% (±8%) or 48% (±7%) for patients with MRD <10(-3). Conventional intensive consolidation treatment reduced MRD to <10(-3) before HSCT in 63% of patients, whereas MRD remained high or increased in the rest of this patient group. Our data support that MRD after induction treatment can be used to quantify the activity of different induction treatment strategies in phase II trials. MRD persistence at ⩾10(-3) before HSCT reflects a disease highly resistant to conventional intensive chemotherapy and requiring prospective controlled investigation of new treatment strategies and drugs.

Critical Situations in Children, Adolescents and Young Adults with Terminal Cancer within the Home Setting.

BACKGROUND: Over the course of terminal cancer towards the end-of-life, children may experience symptoms that lead to distressing critical situations (CS) for the child and caregivers. METHODS: We analysed the records of 133 children cared for by our paediatric palliative care team (PPCT) from 01/98-12/09. A CS was defined as deterioration of a condition caused by a symptom, which was life-threatening or acutely scaring the patient (pt) or caregivers. RESULTS: The majority of pts who died sustained no CS. In 38 (28.6%) pts 45 CS occurred. These accumulated towards the end-of-life (62.2% within the last week). About two-thirds were anticipated. There was no clustering of CS during the night/weekend. Leading symptoms were neurological. In 4 CS a pre-hospital emergency physician was alerted. 5 pts were readmitted to hospital. Most CS (88.9%) could be controlled in the home setting. DISCUSSION: Despite anticipation, a relevant number of pts developed CS, which needed either additional medical intervention or other support by the PPCT. Considering the distressing and suffering character of status epilepticus and dyspnoea, it is important to thoroughly address these conditions in palliative care. CONCLUSION: Advanced planning, close contact, good communication, detailed parental information, and a 24-h on-call service can reduce CS in children with terminal cancer. CS are mainly manageable within the home setting. Treatment of CS should focus on the child's symptoms and wishes, and the needs of the whole family.

Constitutional mismatch repair-deficiency and whole-exome sequencing as the means of the rapid detection of the causative MSH6 defect.

BACKGROUND: Cases of children with more than one type of cancer either diagnosed simultaneously or successively, rarely occur in pediatric oncology. A second malignant neoplasm may be caused by mutagenic effects of the treatment of the primary malignancy and/or may point towards an underlying genetic cancer susceptibility syndrome. One example of such a syndrome is constitutional mismatch repair-deficiency, (CMMR-D) which carries an increased risk of various tumors including childhood hematologic malignancies and Lynch syndrome associated tumors. Timely diagnosis of CMMR-D is crucial, since this diagnosis has implications for the entire family. PATIENT: We report the case of a 15-year-old girl who was born to consanguineous parents. At the age of 20 months she was diagnosed with a T-cell non-Hodgkin lymphoma. Treatment was given according to NHL-BFM 95. 12 years later, an invasive adenocarcinoma of the colon was surgically removed which relapsed shortly afterwards. METHODS: Whole-exome sequencing of germline DNA was employed to rapidly detect the underlying mutation in this suspected CMMR-D patient. RESULTS: After a short turnaround time of less than 3 weeks, the diagnosis of CMMR-D could be confirmed by the identification of a homozygous 29-bp deletion in MSH6 (exon 6), which was confirmed by independent methods. CONCLUSIONS: We demonstrate that "bed-side" whole-exome sequencing is both feasible and cost-effective and may be the method of choice to rapidly uncover the genetical basis of (inherited) diseases.

[Germline mutations in children with cancer - leopoldina meeting report, september 26, 2014, jesuit monastery merzhausen]. / Keimbahnmutationen bei krebskranken Kindern - Symposium der Leopoldina am 26.09.2014 im Jesuitenkloster Merzhausen bei Freiburg.

Under the umbrella of the Leopoldina, National Academy of Science Germany, a one-day workshop took place with experts from Pediatric Oncology, Human Genetics, Jurisprudence and Science Ethics. Professor Dr. Matthias Brandis, former head of the Clinic of Pediatrics and Adolescent Medicine, Albert-Ludwigs-University Freiburg, encouraged the authors to organize this workshop near Freiburg and provided professional and logistic support. Professor Dr. Matthias Brandis serves as the chairmen of our pediatric-gynecological section within the Leopoldina.

[Palliative home care of children: an opinion survey of orphaned parents]. / Häusliche Palliativversorgung von Kindern: Eine Meinungsumfrage bei verwaisten Eltern nach unheilbarer onkologischer Erkrankung.

INTRODUCTION: In Germany, 500 children die of malignancies per year. Many families wish to be cared for in a home setting at the end-of-life. METHODS: Families of children who were cared for by the paediatric palliative care team (PPCT) in a home setting between 01.02.2003 to 30.09.2009 were included in the survey. The questionnaire consisted of 87 items with nominal scaled variables and numeric rating scales (NRS; 1-4, lowest to highest satisfaction) as response options. RESULTS: 84 relatives of 49 children participated (response rate 53.2%). Duration of care varied between 3-246 days. All 49 patients died at home. 98.8% of the respondents were satisfied with their decision for home care. The symptoms pain (86.9%) and fatigue (85.7%) were reported most frequently. Satisfaction with symptom control was high (NRS 3.55±0.49). The respondents were satisfied with communication (NRS 3.73±0.57) and end-of-life care (NRS 3.85±0.90). Satisfaction with psychosocial care (NRS 3.24±0.87) was significantly lower (p<0.05). Parents who stayed in contact with the PPCT by phone and in person were more satisfied with aftercare. DISCUSSION: From parental view satisfying home-care of children with cancer is feasible. Symptom control succeeds in a home setting.

Osteonecrosis in paediatric patients with acute lymphoblastic leukaemia treated on Co-ALL-07-03 trial: a single centre analysis.

BACKGROUND: Osteonecroses (ON) are a serious problem after anti-leukaemic treatment in childhood and critically depend on treatment intensity. We analysed ON incidence, risk factors and outcome in patients (pts) from our institution treated according to the CoALL 07-03 trial. METHODS: Between 01.09.2003 and 31.12.2009, 124 children aged 1-18 years were treated, 22 pts with ON (ARCO I-IV) were assessed by retrospective chart review. Follow-up data were collected as of March 2013. RESULTS: 5-year cumulative incidence of ON grade I-IV was 25%. Median age at ALL diagnosis with vs. without ON was 11 years vs. 4.4 years. In logistic multivariate regression analysis, age was the only independent risk factor for ON (p<0.01). 90.9% of the pts with ON presented with ≥2 bilaterally affected joints, most frequent the weight-bearing joints (95.5%). 77.2% developed ON ≥°III acc. to ARCO. 36.4% underwent core decompression, one patient bilateral total hip arthroplasty. As of March 2013, 12 pts still presented with ON-induced symptoms. DISCUSSION: Our data suggest an overall high incidence of ON in pts treated according to trial CoALL 07-03. Cumulative steroid dose in trial CoALL 07-03 was small, thus, the high CI might be triggered by other treatment-related and study population based risk factors. CONCLUSION: ON are a serious problem concerning long-term sequelae with major impact on activities of daily living. Further prospective evaluation is urgently needed to develop risk-adapted diagnostic strategies and preventive and interventional approaches for high-risk pts.

Efficacy of cyclin-dependent-kinase 9 inhibitors in a murine model of mixed-lineage leukemia.

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

The impact of TEL-AML1 (ETV6-RUNX1) expression in precursor B cells and implications for leukaemia using three different genome-wide screening methods.

The reciprocal translocation t(12;21)(p13;q22), the most common structural genomic alteration in B-cell precursor acute lymphoblastic leukaemia in children, results in a chimeric transcription factor TEL-AML1 (ETV6-RUNX1). We identified directly and indirectly regulated target genes utilizing an inducible TEL-AML1 system derived from the murine pro B-cell line BA/F3 and a monoclonal antibody directed against TEL-AML1. By integration of promoter binding identified with chromatin immunoprecipitation (ChIP)-on-chip, gene expression and protein output through microarray technology and stable labelling of amino acids in cell culture, we identified 217 directly and 118 indirectly regulated targets of the TEL-AML1 fusion protein. Directly, but not indirectly, regulated promoters were enriched in AML1-binding sites. The majority of promoter regions were specific for the fusion protein and not bound by native AML1 or TEL. Comparison with gene expression profiles from TEL-AML1-positive patients identified 56 concordantly misregulated genes with negative effects on proliferation and cellular transport mechanisms and positive effects on cellular migration, and stress responses including immunological responses. In summary, this work for the first time gives a comprehensive insight into how TEL-AML1 expression may directly and indirectly contribute to alter cells to become prone for leukemic transformation.

Cytogenetic aspects of childhood leukemias.

Recurrent non-random chromosome abnormalities, including numerical or structural changes such as translocations, inversions, insertions or deletions within the leukemia cell nucleus, have been discovered in approximately 80% of patients with a malignant hematological disease. These reciprocal translocations correlate with specific cellular subtypes of hematopoeisis at the stage of their maturation arrest and are therefore important for diagnosis. Some of these aberrations are independent prognostic indicators and help to stratify patients into different risk-adapted therapy groups. Owing to new laboratory methods such as the fluorescence in situ hybridization (FISH) and modified polymerase chain reaction (RT-PCR) the chromosomal breakpoints can be investigated and the rearrangements of genes which produce the abnormal proteins can be identified. Due to the high sensitivity of these available data a new prognostic factor, the "minimal residual disease" (MRD), can be investigated at diagnosis and at intervals during the treatment period. Since we now know which oncoproteins are involved, a target-directed therapy with inhibitors might be possible in the future.Standard cytogenetic and molecular genetic analysis of the leukemia karyotype is of the utmost importance for classification (WHO), therapy and therapy results in the acute childhood leukemias.

Allo-SCT using BU, CY and melphalan for children with AML in second CR.

Based on the results from the AML-BFM 98 trial, hematopoietic SCT (HSCT) is recommended for children with AML in second CR only. Here, we retrospectively analyze interphase data of children who underwent HSCT after myeloablative conditioning with BU, CY, and melphalan (BuCyMel) for AML in second remission (CR2) between 1998 and 2009. Out of 152 children, transplant data were available on 109 individuals. Sixty out of 109 children (55%) received BuCyMel. Median age at HSCT was 12.2 years (range 3.0; 18.3). GVHD prophylaxis mostly consisted of CsA and short term MTX with or without antithymocyte globulin. Matched-sibling donors were used for 6/60 analyzed recipients, the remainder either received grafts from matched unrelated (30/60) or mismatched donors. OS after 5 years was 62% (s.e. 6%), relapse incidence 35% (18/60 children) and treatment-related mortality accounted for 12% (7/60) of fatal events. In conclusion, even taking into account possible selection bias in this retrospective analysis, HSCT in CR2 using BuCyMel resulted in a respectable OS. Based on this data the prospective, controlled and centrally monitored AML SCT-BFM 2007 trial has started to recruit patients in January 2010 aiming to generate valid outcome data for further strategy decisions.

Evaluation of the prognostic meaning of C-reactive protein (CRP) in children and adolescents with classical Hodgkin's lymphoma (HL).

BACKGROUND: In adult cancer patients the negative predictive value of elevated CRP levels has been described for several malignancies. Only few studies have analyzed the prognostic role of CRP in children and adolescents with classical HL. In these studies elevated CRP levels correlate with the presence of classical risk factors and adverse outcome. PATIENTS AND METHODS: The prognostic role of CRP for patients with classical HL admitted to the GPOH-HD-2002 study was analyzed retrospectively. RESULTS: CRP levels were documented for 369 of 573 patients. Significant (p<0.05) increased median CRP levels were found in the presence of B-Symptoms (25.7 vs. 5.1 mg/l), extranodal involvement (21.5 vs. 7.5 mg/l), elevated erythrocyte sedimentation rate (ESR, 13.0 vs. 1.0 mg/l) and stage III/IV disease (15.5 vs. 5.3 mg/l). 83.9% of patients with elevated and 45.8% of patients with normal CRP had an ESR >30 mm/h. CONCLUSION: Elevated CRP levels were associated with classical risk factors of HL. CRP and ESR may reflect different biological processes. CRP was prognostic within early stage TG-1 patients treated with reduced treatment, but not within advanced stage TG-2+3.

Efficient control of pandemic 2009 H1N1 virus infection with intravenous zanamivir despite the lack of immune function.

A teenager who acquired 2009 H1N1 influenza A lower respiratory tract infection during total bone marrow and lymphoid aplasia, in the setting of human leukocyte antigen-haploidentical hematopoietic stem cell transplantation, was successfully treated with intravenous zanamivir. This case demonstrates efficient control of pandemic influenza infection by intravenous zanamivir in the absence of any functional immune system, thus suggesting profound antiviral activity.