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Patient-derived xenograft (PDX) models have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, despite increasingly improved methodology, engraftment efficiencies frequently remain low. Humanized three-dimensional scaffold models (ossicle xenotransplantation models) in immunocompromised mice have recently been found to enable improved engraftment rates of healthy and malignant human hematopoiesis. We therefore interrogated the feasibility of using four different three-dimensional ossicle-based PDX models for application with primary MDS samples. In a fully standardized comparison, we evaluated scaffold materials such as Gelfoam, extracellular matrix (ECM), and human or xenogenous bone substance in comparison to intrafemoral (IF) co-injection of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) and CD34

Central venous catheters (CVC) placed either via the internal jugular vein (IJV) or the subclavian vein (SCV) are routinely used in patients with hematologic malignancies. In this retrospective study, we systematically compared CVC-associated complications for both insertion sites, IJV and SCV. Between January 2011 and June 2013, all consecutive patients (n = 87) were included with at least one CVC (n = 153; n = 94 IJV; n = 59 SCV) at our institution due to induction/consolidation for AML/ALL or autologous hematopoietic cell transplantation (HCT). Primary study endpoints were central line-associated (CLABSI), catheter-related (CRBSI) blood stream infections and local inflammation (LI) at the insertion site. CRBSI occurred earlier and more frequently in the IJV- versus the SCV-group with an incidence rate of CRBSI at day 15 of 10% versus 0% (p = .04) and a rate of CRBSI per 1000 CVC days of 5.7 versus 1.2. In addition, CLABSI was detected more often in IJV- compared to SCV-CVC (26% vs. 8%, p = .009). Conversely, LI occurred more frequently and earlier in SCV- versus IJV-CVC (88% vs. 56%, p .0001) with a median time to LI of 9 versus 14 days (p .0001). The strongest risk factor for the endpoints CRBSI, CLABSI, and LI was the insertion site. However, SCV insertion was a risk factor for LI (p = .001, HR: 2.0), insertion in the IJV a risk factor for CLABSI (p = .044, HR: 2.7) and CRBSI (p = .036, HR: 5.4). These results demonstrate a differential effect of the insertion site of CVC in neutropenic patients with a significantly reduced frequency of CVC-related blood stream infections in SCV-CVC.

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a distinct subtype of MDS with quite favorable prognosis and excellent response to treatment with lenalidomide. Still, a relevant percentage of patients do not respond to lenalidomide and even experience progression to acute myeloid leukemia (AML). In this study, we aimed to investigate whether global DNA methylation patterns could predict response to lenalidomide. Genome-wide DNA methylation analysis using Illumina 450k methylation arrays was performed on n=51 patients with MDS del5q who were uniformly treated with lenalidomide in a prospective multicenter trial of the German MDS study group. To study potential direct effects of lenalidomide on DNA methylation, 17 paired samples pre- and post-treatment were analyzed. Our results revealed no relevant effect of lenalidomide on methylation status. Furthermore, methylation patterns prior to therapy could not predict lenalidomide response. However, methylation clustering identified a group of patients with a trend towards inferior overall survival. These patients showed hypermethylation of several interesting target genes, including genes of relevant signaling pathways, potentially indicating the evaluation of novel therapeutic targets. Supplementary Information The online version contains supplementary material available at 10.1007/s00277-021-04492-1.

Context Prediction of AH is frequently undertaken in the clinical setting. The commonly used methods are based on the assessment of skeletal maturation. Predictive algorithms generated by machine learning, which can already automatically drive cars and recognize spoken language, are the keys to unlocking data that can precisely inform the pediatrician for real-time decision making. Objective To use machine learning (ML) to predict adult height (AH) based on growth measurements until age 6 years. Methods Growth data from 1596 subjects (798 boys) aged 0-20 years from the longitudinal GrowUp 1974 Gothenburg cohort were utilized to train multiple ML regressors. Of these, 100 were used for model comparison, the rest was used for 5-fold cross-validation. The winning model, random forest (RF), was first validated on 684 additional subjects from the 1974 cohort. It was additionally validated using 1890 subjects from the GrowUp 1990 Gothenburg cohort and 145 subjects from the Edinburgh Longitudinal Growth Study cohort. Results RF with 51 regression trees produced the most accurate predictions. The best predicting features were sex and height at age 3.4-6.0 years. Observed and predicted AHs were 173.9 ± 8.9 cm and 173.9 ± 7.7 cm, respectively, with prediction average error of –0.4 ± 4.0 cm. Validation of prediction for 684 GrowUp 1974 children showed prediction accuracy r = 0.87 between predicted and observed AH (R2 = 0.75). When validated on the 1990 Gothenburg and Edinburgh cohorts (completely unseen by the learned RF model), the prediction accuracy was r = 0.88 in both cases (R2 = 0.77). AH in short children was overpredicted and AH in tall children was underpredicted. Prediction absolute error correlated negatively with AH (P Conclusion We show successful, validated ML of AH using growth measurements before age 6 years. The most important features for prediction were sex, and height at age 3.4-6.0. Prediction errors result in over- or underestimates of AH for short and tall subjects, respectively. Prediction by ML can be generalized to other cohorts.

Mutations in the CBL gene were first identified in adults with various myeloid malignancies. Some patients with juvenile myelomonocytic leukemia (JMML) were also noted to harbor mutations in CBL, but were found to have generally less aggressive disease courses compared to patients with other forms of Ras pathway-mutant JMML. Importantly, and in contrast to most reports in adults, the majority of CBL mutations in JMML patients are germline with acquired uniparental disomy occurring in affected marrow cells. Here, we systematically studied a large cohort of 33 JMML patients with CBL mutations and found that this disease is highly diverse in presentation and overall outcome. Moreover, we discovered somatically acquired CBL mutations in 15% of pediatric patients who presented with more aggressive disease. Neither clinical features nor methylation profiling were able to distinguish patients with somatic CBL mutations from those with germline CBL mutations, highlighting the need for germline testing. Overall, we demonstrate that disease courses are quite heterogeneous even among patients with germline CBL mutations. Prospective clinical trials are warranted to find ideal treatment strategies for this diverse cohort of patients.

Although acute promyelocytic leukemia (APL) has evolved to the AML entity with the best prognosis, typical 'early death' (ED) events still account for mortality rates of ∼20% in population-based studies. To investigate this poorly understood issue we performed whole transcriptome analysis of

Central venous catheters (CVCs) are extensively used in patients undergoing allogeneic hematopoietic cell transplantation (HCT). In these patients CVC are placed routinely either via the internal jugular vein (IJV) or the subclavian vein (SCV). Purpose of this study was to systematically analyze complications of CVC at different insertion sites in HCT recipients. In this retrospective analysis, all consecutive patients (n = 56) who received a CVC (n = 101) due to allogeneic HCT at our institution between January 2011 and June 2013 were included. Three-lumen standard, nontunneled CVCs were placed via either the IJV (n = 60; 59%) or the SCV (n = 41; 41%). Study endpoints were time to local inflammation at the insertion site, time to fever, time to a combined endpoint of inflammation and fever, central line-associated bloodstream infection (CLABSI), duration of catheterization, catheter lumen obstruction, deep-vein thrombosis, pneumothorax, and catheter-related death. The median duration of catheterization per CVC was almost identical for the IJV and SCV sites (18 days versus 17 days; P not significant). There were no differences in the frequency of CLABSI, deep-vein thrombosis, pneumothorax, and catheter lumen obstruction between IJV and SCV CVC insertion sites. None of the patients died due to a CVC-related cause. Local inflammation occurred less frequently (48% versus 71%; P = .025) and later (median time to local inflammation, 25 days versus 12 days; P = .01) in IJV CVCs versus SCV CVCs. There was a trend toward a median longer time to the occurrence of fever for IJV CVCs compared with SCV CVCs (20 days versus 13 days; P = .07). In the multivariate analysis, diagnosis of acute leukemia (hazard ratio [HR], 1.696; P = .036), SCV CVC (HR, 1.617; P = .039), and neutropenic CVC-days (HR, 2.477; P = .01) were identified as risk factors for the occurrence of local inflammation or fever. In contrast to earlier studies in patients without hematologic malignancies, these data demonstrate that CVCs placed in the SCV are not superior over IJV CVCs. Moreover, local inflammation occurred earlier and more frequently in patients with an SCV CVC.

We present methods enabling rapid non-uniformity and range walk error correction of 3D flash LiDAR imagers that exhibit electronic crosstalk caused by simultaneously triggering too many detectors. This additional electronic crosstalk is referred to as simultaneous ranging crosstalk noise (SRCN). Using a method in which the 3D flash LiDAR imager views a checkerboard target downrange, the SRCN is largely mitigated. Additionally, processing techniques for computing the non-uniformity correction (NUC) and range walk error correction are described; these include an in-situ thermally compensated dark-frame non-uniformity correction, image processing and filtering techniques for the creation of a photo-response non-uniformity correction, and characterization and correction of the range walk error using data collected across the full focal plane array without the need for sampling or windowing. These methods result in the ability to correct noisy test validation data to a range precision of 8.04 cm and a range accuracy of 1.73 cm and to improve the signal-to-noise ratio of the intensity return by 15 to 49 dB. Visualization of a 3D scene corrected by this process is additionally presented.

This paper presents the expansion of dark-frame non-uniformity correction (DFNUC) techniques to include compensation for thermal drift in a 128×128 PIN diode 3D flash LiDAR camera. Flash LiDAR cameras are operated in various climates, which makes thermal compensation necessary in the dark NUC algorithm. The thermal excitation of electrons has a significant effect on the dark current in an InGaAs PIN photodetector and on the CMOS readout circuitry, thus impacting the output image. This is a well-known phenomenon in imaging sensors and various algorithms have been established to address thermal drift. This paper adapts a linear model for dark signal calibration used in infrared cameras for the calibration of a PIN diode flash LiDAR camera in both intensity and range return. The experimental process involves collecting dark frames in increments of the internal camera temperature from 22°C to 36°C using thermoelectric (TE) cooling modules. A linear trendline is developed for each individual pixel based on the average frame return, which suppresses the random temporal noise and isolates the dark signal return. The trendline helps form a model for the dark frame offset as a function of temperature, which is used for the dark-frame NUC process. The dark-frame NUC with thermal drift compensation is then evaluated by correcting dark frames at various operating temperatures. Finally, illuminated scenes captured by the camera with a 5.91ns, 842.4μJ pulsed laser at 5Hz are corrected at multiple operation temperatures to show the effectiveness of the dark non-uniformity correction algorithm.

We present experimental methods and results for photo-response non-uniformity correction (PRNUC) in range for a 3D flash LiDAR camera from non-optimal static-scene calibration data. Range walk is also corrected. This method breaks up the camera’s focal plane array (FPA) into 16 × 16 windowed regions of interest that are incrementally captured and stitched together in post-processing across the entire FPA. The illumination was not uniform, thus requiring additional methods described by our paper to create an acceptable correction. We present the results from a full non-uniformity correction and range walk error correction processed for a set of independently collected validation frames; these validation frames used identical experimental conditions and the same target as was collected for the corrections. We will show that this experimental approach improves range accuracy and range precision of the corrected validation frames despite the sub-optimal conditions of the data used to compute the corrections; the single shot range precision is corrected to 33 cm, as compared to a modeled precision of 15.65 cm, while the accuracy is corrected to 252 cm. This method has implications for simplification of characterization of non-uniformity and range walk error, and its subsequent correction, in 3D flash LiDAR cameras.

BackgroundDespite the intensity of hematopoietic stem cell transplantation (HCT), relapse remains the most common cause of death in juvenile myelomonocytic leukemia (JMML). In contrast to other leukemias where therapy is used to reduce leukemic burden prior to transplant, many patients with JMML proceed directly to HCT with active disease. The objective of this study was to elucidate whether pre-HCT therapy has an effect on the molecular burden of disease and how this affects outcome post-HCT.ProcedureTwenty-one patients with JMML who received pre-HCT therapy and were transplanted at UCSF were analyzed in this study. The mutant allele frequency of the driver mutation was assessed before and after pre-HCT therapy, using custom amplicon next-generation sequencing.ResultsOf the 21 patients, seven patients (33%) responded to therapy with a significant reduction in their mutant allele frequency and were classified as molecular responders. Six of these patients received moderate-intensity chemotherapy, one patient received only azacitidine. The 5-year progression-free survival after HCT of molecular responders was 100%versus 61% for nonresponders (P=.12). Survival of molecular nonresponders was not improved by use of high-intensity conditioning, but patients were salvaged if they experienced severe graft versus host disease. There were no baseline clinical characteristics that were associated with response to pre-HCT therapy.ConclusionsDespite the myelodysplastic nature of JMML, patients treated with pre-HCT therapy can achieve molecular remissions. These patients experienced a trend toward improved outcomes post-HCT. Importantly, molecular testing can be helpful to distinguish between responders and nonresponders and should become an integral part of clinical care.

Objectives Randomized comparison of two treatment strategies in frontline therapy of acute promyelocytic leukemia (APL): all-trans retinoic acid (ATRA) and double induction intensified by high-dose cytosine arabinoside (HD ara-C) (German AMLCG) and therapy with ATRA and anthracyclines (Spanish PETHEMA, LPA99). Patients and results Eighty of 87 adult patients with genetically confirmed APL of all risk groups were eligible. The outcome of both arms was similar: AMLCG vs PETHEMA: hematological complete remission 87% vs 83%, early death 13% vs 17% (P = .76), overall survival, event-free survival, leukemia-free survival, cumulative incidence of relapse at 6 years 75% vs 78% (P = .92); 75% vs 68% (P = .29); 86% vs 81% (P = .28); and 0% vs 12% (P = .04, no relapse vs four relapses), respectively. The median time to achieve molecular remission (RT-PCR negativity of PML-RARA) was 60 days in both arms (P = .12). The AMLCG regimen was associated with a longer duration of neutropenia (P = .02) and a higher rate of WHO grade ≥3 infections. Conclusions The small number of patients limits the reliability of conclusions. With these restrictions, the outcomes of both approaches were similar and show the limitations of ATRA and chemotherapy. The HD ara-C-containing regimen was associated with a lower relapse rate in high-risk APL.

Anamnese, Untersuchungen und Diagnose Eine junge Patientin stellte sich wegen spontaner Hamatome im Krankenhaus vor. Aufgrund der Kombination von Panzytopenie und Koagulopathie im Blut sowie den beweisenden zyto- und molekulargenetischen Untersuchungen des Knochenmarks wurde eine Niedrigrisiko akute Promyelozytenleukamie (APL) diagnostiziert. Therapie und Verlauf Unter der Kombinationstherapie mit ATRA und ATO entwickelte die Patientin ein schweres Differenzierungssyndrom bei Hyperleukozytose. Dies konnte nur durch klinische Expertise und den massiven Ersatz von Blutprodukten und Gerinnungsfaktoren stabilisiert werden. Folgerung Der Fall verdeutlicht die Schwierigkeiten und Gefahren der APL-Induktionstherapie auch bei gunstiger Ausgangslage trotz neuer, insgesamt risikoarmer Therapien.

To date risk stratification in acute promyelocytic leukemia (APL) is based on highly dynamic leukocyte and platelet counts only. To identify a more robust risk stratification model, a molecular risk score was developed based on expression levels of the genes BAALC, ERG and WT1. Hereby, the main focus was on prediction of relapse. The integrative risk score divided patients into two groups with highly significant differences in outcome. It discriminated a high risk group with a high incidence of relapse successfully from a low risk group with no APL-related events after achievement of first remission. Especially the concurrent presence of molecular risk factors showed to be a negative prognostic factor in APL. The molecular risk score might be a promising approach to guide monitoring of APL patients and therapeutic decisions in the future.

Wilms' tumor 1 gene (WT1) is known to be highly expressed in acute promyelocytic leukemia (APL) but information on its impact on prognosis is lacking. WT1 expression was analyzed in bone marrow samples of 79 patients with APL at initial diagnosis. Patients had a differing outcome according to their level of WT1 expression. In patients who achieved a complete remission (CR), low or high WT1 expression was significantly associated with inferior overall survival (OS) compared to intermediate WT1 expression (49% for WT1high vs. 63% for WT1low vs. 93% for WT1int; p=0.008). Moreover, there were significant differences in relapse-free survival (RFS) between the three expression groups (42% for WT1high vs. 63% for WT1low vs. 83% for WT1int; p=0.047). In multivariable analysis WT1 expression showed an independent prognostic impact on OS of responders to induction therapy. In conclusion, the level of WT1 expression can add prognostic information in APL risk stratification.

Introduction Although treatment of acute promyelocytic leukemia (APL) has evolved dramatically during the past decades, especially with the introduction of all-trans retinoic acid, risk stratification remains an important issue. To date, relapse risk can be predicted by leukocyte and platelet counts only. In the present report, we present a validation study on 3 candidate genes and a newly developed molecular risk score for APL in 2 independent patient cohorts. Patients and Methods An integrative risk score combining the expression levels of BAALC , ERG , and WT1 was calculated for 79 de novo APL patients from the original cohort and 76 de novo APL patients from a validation cohort. Gene expression analysis was executed the same for both cohorts, and the results regarding the effect on patient outcomes were compared. Results The expression levels of BAALC , ERG , and WT1 were similar in both cohorts compared with the healthy controls. The relapse and survival rates were not different between the low- and high-risk patients according to the Sanz score. However, application of the molecular risk score on the validation cohort distinctly discriminated patients according to their risk of relapse and death just as in the original APL cohort, although single gene analyses could not reproduce the negative prognostic impact. Conclusion The analysis clearly validated the prognostic effect of the integrative risk score on the outcome in APL patients. The value was further empowered because the single gene analyses did not show similar results. Whether the integrative risk score retains its prognostic power in the chemotherapy-free setting should be investigated further.

In acute promyelocytic leukemia (APL), relapse occurs in about 15 % of cases and is a major cause for death. Molecular markers identifying patients at high risk for relapse are not well established. High expression of the transcription factor Ets-related gene (ERG) is associated with inferior overall survival (OS) and disease-free survival in different types of hematologic malignancies. There are no data available about the impact of ERG expression in APL. ERG expression levels were analyzed in bone marrow samples of 86 APL patients at initial diagnosis. High ERG expression was significantly associated with an inferior OS in patients who had reached first complete remission. It was also significantly correlated with inferior relapse-free survival (RFS) and time to relapse (i.e., relapse-free interval, RFI). In multivariate analysis, high ERG expression had an independent negative impact on RFS and RFI. High ERG expression was significantly associated with inferior OS, RFS, and RFI. Moreover, in multivariate analysis, it maintained its value as an independent negative prognostic factor with regard to RFS and RFI. Therefore, ERG expression might serve as a molecular marker for risk stratification in APL and might identify patients who could benefit from intensified treatment regimens.

Introduction: In B-cell leukemia and lymphoma, leukemia initiating cells occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B-cell development (Le Viseur 2008). Unlike stem cells, where self-renewal is regulated by a developmental hierarchy, in the B-cell lineage, positive selection events, i.e. induced by antigen-receptor (BCR) signaling dictates their ability to self-renew. Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple epithelial tissues, however the role of Lgr5 in hematopoietic cells was not explored. Results: Upon successful completion of immunoglobulin V(D)J gene recombination and first encounter of antigen represent key events in the life of a B-cell that promote survival and positive selection. Here, we found that both events result in upregulation of Lgr5 expression in B cell precursors in the bone marrow and germinal center B cells. Likewise, engagement of BCR signaling on B-cell lymphomas and oncogenic BCR-signaling mimics in leukemia strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway.In patients with B-cell leukemia, higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. Inducible ablation of Lgr5 during earliest stages of B-cell development resulted in a >100-fold reduction of absolute B-cell numbers. Studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear β-catenin and increased expression of β -catenin target genes. Deletion of Lgr5 abolished colony forming capacity and reduced the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of β-catenin resulted in rapid cell death in normal and malignant B cells. Conclusion: Lgr5-expression and positive B-cell selection is induced by BCR-engagement by antigen or oncogenic mimics of BCR signaling in B-cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear β-catenin and enables B-cell survival and transformation through negative regulation of Wnt-signaling. Targeting Lgr5 using a novel Lgr5-ADC seems promising to deplete B-cell leukemia- and lymphoma-initiating cells. Citation Format: Kadriye Nehir Cosgun, Anna Hecht, Xin Yang, Maurizio Mangolini,, Ali Aghajanirefah, Gang Xiao, Teresa Sadras, Zhengshan Chen, Lars Klemm, Huimin Geng, Chao Hong, Qingxiao Song, Deye Zeng, Hassan Jumaa, Defu Zeng, Hans Clevers, Markus Muschen. Lgr5 mediates positive B-cell selection and is critical for initiation and survival of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4515.

Background. Chronic active BCR or TCR signaling or its oncogenic mimics result in nuclear accumulation of NF-kB. Oncogenic mimicry of BCR- and TCR signaling can be induced by viral oncoproteins (e.g. LMP2A (EBV), K1 (KSHV) and Tax (HTLV1), activating mutations in the CD79A, CD79B and CD3Z signaling chains or genetic lesions driving tyrosine kinase (e.g. BCR-ABL1), ERK (RAS, BRAF, PLCG1) or PI3K (PIK3CA, PIK3R1) signaling. While BCR and TCR signaling induces positive selection, survival and proliferation in normal lymphocytes, the majority of B cell and T cell malignancies are driven by oncogenic activation of this pathway. Concept & central hypothesis. Oncogenic drivers in B- and T-lymphoid malignancies function as mimics of B- and T-cell receptor (BCR or TCR) signaling. Oncogenic activation of BCR- or TCR-signaling represents the functional equivalent of positive selection during normal lymphocyte development. Addiction to survival and proliferation signals (or the equivalent of positive selection) is a common feature in many types of cancer. However, B- and T-lymphoid malignancies are unique in that they are also subject to an active negative selection process. B- and T-cells expressing autoreactive antibodies and TCRs, respectively, can cause systemic autoimmunity. As a safeguard against autoimmune diseases, lymphocyte development evolved autoimmunity checkpoints (AIC) to eliminate autoreactive clones . Owing to negative selection of autoreactive B- and T-cells through AIC activation, lymphoid cells fundamentally differ in their signaling requirements from other cell types. Four recent studies from our group showed that despite malignant transformation, B cell- and T cell-lineage leukemia and lymphoma cells are fully sensitive to negative selection and AIC-activation resulting (Chen et al., Nature 2015; Shojaee et al., Cancer Cell 2015; Shojaee et al., Nature Med 2016; Chan et al., Nature 2017). Results: AIC-activation in various lymphoid malignancies is achievable by pharmacological hyperactivation of BCR- and TCR-signaling above a maximum threshold, e.g. pharmacological inhibition of PTEN, SHIP1 or DUSP6 phosphatases (Figure). Studying genetic models of B cell malignancies, we observed that Cre-mediated deletion of Pten, Ship1 and constitutive deletion of Dusp6 caused hyperactivation of SYK, PI3K and ERK-pathways, leading to AIC-activation. Unlike other types of cancer, B- and T-cell malignancies are uniquely susceptible to clonal deletion induced by hyperactive signaling from an autoreactive BCR or TCR. Hence, targeted AIC-activation can be leveraged for eradication of drug-resistant leukemia and lymphoma clones. Studying engineered B cells for inducible activation of autoreactive BCR-signaling, we discovered that targeted hyperactivation of SYK, PI3K and ERK in B cell malignancies represents the functional equivalent of an autoimmunity checkpoint (AIC) for elimination of autoreactive clones. Likewise, transformed B cell tumors are uniquely vulnerable to AIC activation, suggesting that targeted activation of this checkpoint represents a novel strategy to induce cell death in otherwise drug-resistant B cell malignancies. Conclusion: Normal B- and T-cells are positively selected for BCR or TCR signaling of intermediate strength (moderate activation of SYK, PI3K and ERK). In the absence of a functional BCR/TCR, SYK, PI3K and ERK activity fall below a minimum threshold, resulting in death by neglect. Hyperactivation above maximum thresholds (e.g. autoreactive BCR/TCR) triggers negative selection and cell death via AIC-activation. Targeted therapy of cancer typically focuses onagents that suppress oncogenic signaling below a minimum threshold . Our results support a novel strategy to overcome drug-resistance in B- and T-cell malignancies based on targeted activation of autoimmunity checkpoints (AIC) for removal of autoreactive cells. Figure Figure. Disclosures Muschen: Pfizer: Research Funding; AbbVie: Research Funding.

Ein 24-jahriger, HIV-positiver Patient wurde wegen progredienter Zephalgien stationar aufgenommen. Bei Aufnahme wurde ein adaquat orientierter Patient mit starksten holozephalen Kopfschmerzen, Ubelkeit und Erbrechen sowie diskretem Meningismus ohne neurologische Ausfalle gesehen. In der Lumbalpunktion konnten Kryptokokken direkt nachgewiesen werden. Der Patient wurde mit einer Dreifachkombination aus Amphotericin B, Flucytosin und Fluconazol behandelt, und zeitnah wurde eine antiretrovirale Therapie (ART) bei schwerer Immundefizienz begonnen. Der Allgemeinzustand besserte sich kontinuierlich, Kryptokokken waren nicht mehr nachweisbar und es wurde eine Erhaltungstherapie mit Fluco nazol durchgefuhrt. Etwa 2 Monate spater traten wieder starkste Kopfschmerzen auf. In der Liquorpunktion wurden erneut massenhaft Kryptokokken nachgewiesen. Die Resistenztestung der Kryptokokken ergab eine intermediare Resistenz gegenuber Fluconazol. Daraufhin wurde Fluconazol durch Voriconazol ersetzt. Hierunter war der Patient zunachst anhaltend beschwerdefrei. 8 Monate nach Beginn der ART kam es erneut zu massiven Kopfschmerzen und Erbrechen. Magnetresonanztomographisch war ein diskretes Hirnodem nachweisbar. Hinweise auf eine opportunistische Infektion oder ein Lymphom ergaben sich nicht; wegen des daher vermuteten Immunrekonstitutionssyndroms (IRIS) wurden Kortikosteroide appliziert. Hierunter kam es rasch zu einem volligen Sistieren samtlicher Beschwerden. Der Patient ist seither (19 Monate nach Erstdiagnose) beschwerdefrei. Die Kryptokokkenmeningoenzephalitis gehort zu den HIV-assoziierten Erkrankungen. Therapeutisch sollte eine antimykotische Kombinationstherapie durchgefuhrt werden. Allerdings muss auch bei Kryptokokken an Resistenzen gedacht werden. Wie bei anderen Infektionen kann es auch bei einer Kryptokokkenmeningoenzephalitis zu einem IRIS kommen.

Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on clinical parameters, namely leukocyte and platelet counts at initial diagnosis as combined in the Sanz Score. However, during the last years the influence of additional molecular genetic markers on prognosis of APL patients has been postulated. In 2015 we published the results of a molecular risk score integrating expression data of the genes brain and acute leukemia, cytoplasmic (BAALC), ets' related gene (ERG) and Wilms' Tumor 1 (WT1) with strong independent influence on outcome and relapse risk of APL patients treated in the German AMLCG studies (Hecht et al., Leuk Res 2015). The aim of our study was to validate our data in an independent patient cohort. Methods: In cooperation with the German SAL (Study Alliance Leukemia) group we obtained a validation set of samples of mononuclear cells derived from the bone marrow of 76 patients with confirmed diagnosis of APL prior to therapy. The validation cohort consisted of 37 female and 39 male patients with a median age of 50 years (range 20-82 years; Table 1). Patients were diagnosed and treated between 2000 and 2014 mostly with an ATRA plus Idarubicin based induction therapy followed by Sanz score-dependent consolidation and maintenance chemotherapy. RNA was extracted using the AllPrep DNA/RNA Mini Kit and cDNA was synthesized using 500µg of RNA with a QuantiTect Reverse Transcription Kit (both Qiagen, Hilden, Germany). Expression levels of BAALC, ERG and WT1 were then analyzed using quantitative real-time RT-PCR with the same conditions and primers as used in the original test cohort and the same calibrator cDNA from cell lines was used for the analysis. The following gene expression levels were defined as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression ≥75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low or high). The integrative risk score was calculated as described before: For the presence of one of the mentioned risk factors, one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors). Overall survival (OS), relapse free survival (RFS) and cumulative incidence of relapse (CIR) were calculated using the Kaplan-Meier method. Results: The expression of the three genes BAALC, ERG and WT1 compared to healthy controls were exactly the same as in the original cohort. Application of the molecular risk score on APL patients of the validation cohort clearly discriminated patients according to their risk comparable to the original APL cohort (Figure 1). Patients with 0 points had a very good prognosis with no deaths or relapses, whereas patients with 3 points (i.e. concurrent presence of all three risk factors) had a very poor outcome with an OS of 51%, a RFS of 50% and a CIR of 38%. The differences between patients with 1 and 2 points were less pronounced in the validation cohort. However, the statistical analyses in the validation cohort did not yield significance. As the main reason we assume that the validation cohort altogether showed a significantly improved OS and RFS compared to the original cohort which was treated a decade earlier, so differences between the subgroups could not be that pronounced. For both cohorts stratification by the Sanz Score did not show significant differences in outcome (analysis for RFS p=0.46 in original cohort and p=0.44 in validation cohort). Conclusion: A validated molecular-based integrative risk score was able to define subgroups of APL patients with different outcome independently of the Sanz score. The validation of the strong influence of the combination of molecular risk factors is particularly interesting as the new analyses of the three genes BAALC, ERG and WT1 separately did not confirm the strong results of the single analyses of each gene. The discrimination of patients according to the risk score is evident though. This argues for their integration in future studies. Figure 1 Comparison of the original cohort and the validation cohort. Discrimination by the integrative risk score (0-3points) for OS, RFS and CIR. Figure 1. Comparison of the original cohort and the validation cohort. Discrimination by the integrative risk score (0-3points) for OS, RFS and CIR. Disclosures No relevant conflicts of interest to declare.

We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leukemia (APL). At 10 years, the overall survival (OS) was 66% in all patients and 75% in patients who achieved a complete remission (CR). Patients in the BAALC(low) group showed an OS of 87% as compared to 60% in the BAALC(high) group (p=0.019). This difference was more pronounced in treatment responders (92% vs. 70%; p=0.035). In multivariate analyses low BAALC expression retained its prognostic relevance. In conclusion, BAALC expression analysis might be useful in further risk stratification in APL patients.

Introduction: Insulin-like growth factor binding proteins (IGFBP) are carriers of insulin-like growth factors (IGFs). They prolong their half-life and modulate their availability and activity. The IGF-signaling system has been shown to have an important role in various solid cancers and hematologic malignancies. High levels of IGFBP2 and IGFBP7 have been associated with chemoresistance, relapse and inferior survival in different leukemias. Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML), which has been reported to have increased expression of IGFBP2. However, there is no data on its impact on prognosis. In addition, no data exist on IGFBP7 in APL. The aim of this study was to elucidate the influence on prognosis of both candidate genes in APL patients. Methods: Expression levels of IGFBP2 und IGFBP7 were retrospectively analysed in bone marrow (BM) samples at the time of initial diagnosis from 69 APL patients (42 female, 27 male) after informed consent. Median age of patients was 46 years (range 19 to 82 years). All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) studies. Treatment consisted of simultaneous ATRA and double induction chemotherapy including high dose ara-C, one cycle of consolidation chemotherapy and 3 years monthly maintenance chemotherapy. In patients older than 60 years, the second induction cycle was at the discretion of the treating physician. Three patients (4%) received an induction of ATRA and an anthracycline without ara-C. BM samples of 22 healthy volunteers served as a control group. Multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) was performed on a LightCycler® 480 (Roche, Mannheim, Germany) PCR system. Glucose-6-phosphat isomerase was used as a housekeeping gene. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used after determination of PCR efficiencies. cDNA from the cell line K562 served as a calibrator in each run. All reactions were performed in triplicates. IGFBP2 expression groups were defined as follows: Patients with IGFBP2 expression below or equal the 25th percentile (IGFBP2low) were compared to patients with higher IGFBP2 expression (IGFBP2high). Overall survival (OS), relapse free survival (RFS) and the cumulative incidence of relapse (CIR) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). Results: Expression levels of IGFBP2 did not differ between APL patients and healthy controls. However, there was a significantly higher relapse rate in APL patients with low IGFBP2 expression (CIR: 25% in the IGFBP2low group vs. 5% in the IGFBP2high group; p=0.04). Accordingly, RFS of patients in the IGFBP2low group was also inferior (41% vs. 81% in the IGFBP2high group; p=0.0002; Fig. 1). The OS of patients who had responded to induction therapy was also influenced by IGFBP2 expression (OS of responders: 61% for IGFBP2low vs. 83% for IGFBP2high; p=0.02). However, there was no significant difference in the analysis of OS of all patients including patients who suffered early death. In contrast to these findings, IGFBP7 was expressed significantly lower in APL patients compared to healthy controls (p Conclusion: Of the two analysed IGFBP family members only IGFBP2 showed impact on the prognosis of APL patients. Remarkably, IGFBP2 was not overexpressed compared to healthy controls in our patient cohort. The reason might be that whole BM samples of healthy controls were used instead of subpopulations. Still, among the APL patients cohort its expression showed a strong influence on prognosis especially on relapse rate and RFS. To find low expression as a negative prognostic marker is surprising as for leukemias only high expression has been reported as a negative factor. However, IGFBP2 has been proposed to suppress tumor development through binding IGFs and preventing IGF-receptor driven tumorigenesis. This is supported by the fact that the IGFBP2 promotor is hypermethylated in various types of cancer. In summary, we identified low IGFBP2 expression as a novel prognostic marker for APL. Further investigations are warranted to clarify its possible role in leukemia. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Central venous catheters (CVCs) are extensively used in patients undergoing hematopoietic cell transplantation (HCT) or induction chemotherapy for acute leukemia. In these patients CVCs are placed routinely either via internal jugular (IJV) or subclavian veins (SCV). Several studies have compared rates of complications between both insertion sites, particularly in the intensive care unit setting. However, to our knowledge there are no data in the literature comparing the insertion sites IJV versus SCV in patients undergoing allogeneic HCT or high dose therapy for hematologic malignancies. Thus, it was the purpose of this study to analyze systematically complications of CVCs at the different insertion sites in these patients. Patients and methods: All consecutive patients who were treated in the unit for adult HCT at our institution from 01/2011 to 06/2013 were included into this retrospective analysis. Inclusion criterion was insertion of a CVC due to allogeneic HCT, autologous HCT or induction therapy for AML or ALL. 3-lumen standard CVCs (Arrow, Reading, PA) were placed after informed consent either via the IJV or the SCV. CVCs were inserted by experienced physicians after disinfection by 70% propanolol under full barrier precautions. The insertion site was chosen at the responsible physician´s own discretion. CVCs were covered by chlorhexidine gluconate impregnated dressings, either as transparent polyurethane (Tegaderm CHG, 3M, Saint Paul, MN) or as sponge dressing (Biopatch, Ethicon, Cincinnati, OH). No patient received a systemic antibiotic prophylaxis. Reasons for premature removal of CVCs were suspected or proven CVC-related blood stream infection (BSI), progression of local infections at the insertion site, or any other CVC related severe adverse event. End points of this study were local infections, fever, BSI, duration of catheterization per CVC, congestion of the CVC, thrombosis, pneumothorax and suspected or proven catheter related deaths. Results: During the study period 255 CVCs were placed in 170 consecutive patients [median age: 59 years (range 19-79), F: n=63; M: n=107] due to allogeneic HCT (65 patients, 101 insertions), autologous HCT (30 patients, 33 insertions) or induction therapy for AML or ALL (75 patients, 121 insertions). Underlying diseases were AML (n=102), ALL (n=9), MDS (n=15), MPN (n=4), lymphoma (n=21) or plasma cell neoplasia (n=19). Out of the 255 CVSs 155 (61%) were sited via the IJV and 100 (39%) via the SCV. Among the 101 CVCs in the allogeneic HCT group 60 (60%) were placed in the IJV and 41 (41%) in the SCV. The median duration of catheterization/per CVC for the entire group was 16 days (range 3-49, IJV: 17, SCV: 15 days, ns). The corresponding data for the allogeneic HCT group were 18 (IJV) and 17 day (SCV). The events fever or local infection occurred in 75% (45/60) of IJV- and in 95% (39/41) of SCV-CVCs (p=0.0084) in the allogeneic HCT group. Moreover, in SCV-CVCs these events arose significantly earlier (median time to event 7 vs. 10 days in the IJV (p=0.012). Focusing only on the event local infection the results were also superior for the IJV-CVCs: 28/60 (47%) vs. 29/41(71%) for SCV-CVCs (p=0.026). For the entire cohort of CVCs data were similar: The events fever or local infection occurred in 127/155 (82%) of IJV- and 95/100 (95%) of SCV-CVCs (p=0.025). Median time to these events were 8 (IJV) vs. 10 days (SCV, p=0.0009). For local infections alone the differences were even more pronounced: 81/155 (IJV: 52%) vs. 81/100 (SCV: 81%, p Conclusions: In contrast to earlier studies on patients without hematologic malignancies these data demonstrate clearly that CVCs placed in the SCV are not superior. Moreover, local infections and fever occurred significantly earlier and more frequent in SCV-CVCs in patients undergoing allogeneic HCT. Disclosures No relevant conflicts of interest to declare.

To search for new copy number alterations (CNAs) in acute promyelocytic leukemia (APL), we analyzed DNA from leukemic blasts of 93 acute promyelocytic leukemia (APL) patients with Genome-Wide SNP 6.0 arrays (SNP-A). We identified 259 CNAs consisting of 170 heterozygous deletions, 82 amplifications, and 7 regions of copy number neutral loss of heterozygosity. One of the most common CNAs was a deletion on chromosomal subband 1q31.3 in 13 of 93 (14%) patients encompassing the coding regions for the microRNAs mir181a1/b1. In multivariable analysis with the covariates age, white blood cell count, platelet count, and FLT3-ITD/FLT3 D835 mutations we found that after adjustment for patients' age (P < 0.0001), patients with 2 or more CNAs detected by SNP-A had a higher risk of death (hazard ratio = 5.942, P = 0.0015) than patients with 0 or 1 CNA. Deletions of 1q31.3 were associated with a higher number of CNAs (median 2 vs. 8, P < 0.0001) and were a strong independent prognostic factor for an increased risk of relapse (hazard ratio = 28.9, P = 0.0031). This study presents a comprehensive assessment of new CNAs as pathomechanistically relevant targets and possible prognostic factors which could refine risk stratification of APL. © 2012 Wiley Periodicals, Inc.

A 24-year-old HIV-positive patient was admitted to hospital on account of increasing headache.On admission, a patient with severe headache, nausea and vomiting but without neurologic deficiencies was seen. The diagnosis of a cryptococcal meningoencephalitis could be confirmed by direct detection of cryptococci in the liquor.The patient was treated with a combination of fluconazole, flucytosine and amphotericin B, and an antiretroviral therapy (ART) was started on account of the severe immunodeficiency. The patient improved during the following weeks, and fluconazole was administered as maintainance therapy. About 2 months later, the patient presented again with severe headache. On lumbar puncture, a great amount of cryptococci could be detected, and the antimycotic combination therapy was restarted. An antimycotic testing of the cryptococci revealed a partial resistance to fluconazole. Therefore, fluconazole was replaced by voriconazole which has been continued ever since. Cryptococci could not be detected on further investigations of the liquor. The patient's condition worsened again 8 months after initiation of the ART. MR scan showed a slight cerebral edema. There was no hint of an opportunistic infection nor of a lymphoma. The complaints were supposed to be due to an immune restoration inflammatory syndrome (IRIS), and administration of high steroid dosages was started. The complaints resolved within 48 h, and the patient's condition has been stable ever since (19 months).Cryptococcal meningoencephalitis is an opportunistic infection in AIDS. Therapeutically, various regimens containing two to three different antimycotic drugs show good efficacy but resistance to antimycotics has to be considered. As in other HIV associated infections, an IRIS has to be taken into account.

Introduction With current therapy regimens over 75% of patients with de novo acute promyelocytic leukemia (APL) can be cured. Approaches to further improve patient outcome by stratifying patients at the time of initial diagnosis according to their individual risk and to adjust therapy accordingly have been based on clinical features only. Molecular markers have not been established for risk stratification as yet. Recently, we have shown that high expression levels of the genes brain and acute leukemia, cytoplasmic (BAALC) and ets related gene (ERG) are associated with inferior outcome in APL patients. In addition, data indicate that aberrant expression of the gene Wilms’ tumor 1 (WT1) is a negative prognostic factor with regard to overall survival (OS) after complete remission (CR) and relapse free survival (RFS) in APL. In this study we evaluated the prognostic relevance of a combined score integrating the expression levels of the above mentioned genes to further improve risk stratification in APL patients. Methods Expression levels of BAALC, ERG and WT1 of 62 patients with newly diagnosed APL were retrospectively analyzed in bone marrow mononuclear cells using multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR). Median age of patients was 47 years (range: 19 to 82y). All patients gave informed consent. Patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study with a treatment of simultaneous ATRA and double induction chemotherapy including high-dose ara-C, consolidation and maintenance chemotherapy. The following gene expression levels were identified as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression >75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low/high). A risk score was developed as follows: for the presence of one of the mentioned risk factors one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low/high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors) could be allocated to one patient. Accordingly, patients were divided into four risk groups: 7 patients scored 0 points (= low risk), 27 patients scored 1 point (= intermediate 1 risk), 19 patients scored 2 points (= intermediate 2 risk) and 9 patients scored 3 points (= high risk). Subsequently, OS, RFS and relapse free interval (RFI) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the four risk groups (p Results The integrative risk score divided patients into four groups with significantly different outcome. The low risk group showed a RFS of 100% at 10 years of follow-up compared to the intermediate 1 risk group with 81%, the intermediate 2 risk group with 58% and the high risk group with a RFS of 42% only (median survival: 4.6y) (p=0.02). In accordance, the RFI differed significantly between the four groups: low risk 100%, intermediate 1 risk 100%, intermediate 2 risk 89% and high risk 71% (p=0.049). There was no statistically significant difference between the 4 groups with regard to OS in the entire patient cohort. However, there was a clear trend towards a difference in OS in patients who achieved a CR after induction therapy: low risk 100%, intermediate 1 risk 81%, intermediate 2 risk 68% and high risk 53% survival at 10 years of follow-up (p=0.09). Conclusion Integration of expression levels of the genes BAALC, ERG and WT1 into a scoring system identifies 4 risk groups with significantly different outcome with regard to RFS and RFI. It might be a promising approach to guide therapeutic decisions in patients with APL. However, multivariate analyses and validation of these data in an independent patient cohort is warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 3531 Introduction: In acute promyelocytic leukemia (APL) the translocation t(15;17) leading to the fusion transcript PML-RARα is necessary but not sufficient to generate leukemia. However, additional molecular alterations leading to overt APL remain elusive. The introduction of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) in the treatment of APL has resulted in long term survival in about 75% of patients with de novo APL, but still, relapse occurs in about 15% of cases. However, markers identifying patients at high risk for relapse are not well established yet. The ets related gene (ERG) is a downstream effector of mitogenic signaling transduction. It is involved in key steps regulating cell proliferation, differentiation and apoptosis. High expression of ERG has been shown to be associated with inferior overall survival (OS) and disease free survival (DFS) in different types of hematologic malignancies such as acute myeloid leukemia and T cell acute lymphoblastic leukemia. The aim of this study was to evaluate the role of ERG expression in APL development and to elucidate its value as a prognostic molecular marker in APL patients. Methods: Bone marrow (BM) mononuclear cells were obtained from 86 APL patients (50 female, 36 male) after informed consent. Median age of patients was 47 years with a range from 19 to 82 years. All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed in triplicates by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) on a LightCycler 480 (Roche, Mannheim, Germany). Glucose-6-phosphat isomerase (GPI) served as a housekeeping gene. The comparative cycle threshold (CT) method was used to determine relative ERG expression levels and the cycle number difference (ΔCT=GPI-ERG) was calculated using the mean of ΔCT from the replicates, that is μ(ΔCT) and expressed as 2μ(ΔCT). cDNA from KG1a cells served as a calibrator in each run. Statistical analyses were performed using SAS version 9.2. ERG expression groups were defined as follows: ERG expression higher than the 75th percentile (ERGhigh) and ERG expression lower than the 75th percentile (ERGlow). Time to complete remission (CR), disease free survival (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the curves. Significance was set at p < 0.05. Results: For patients who achieved CR high ERG expression was significantly associated with an inferior OS as compared to lower ERG expression (49% vs. 80% at 10 years, p=0.02). The effect on DFS was even stronger: only 28% of patients in the ERGhigh group survived without relapse after a follow-up of 10 years as compared to 75% of patients in the ERGlow group (p=0.001). However, the expression of ERG did not have an influence on whether patients achieved CR, neither on how fast they achieved CR. Conclusion: In univariate analysis high ERG expression was significantly associated with inferior outcome concerning overall survival and disease free survival. Therefore, ERG expression might serve as a molecular marker for risk stratification. It might identify patients who could benefit from intensified treatment regimens such as maintenance therapy or allogeneic stem cell transplantation. However, multivariate analyses and validation of these data in an independent patient cohort is warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 420 Introduction: The characteristic hallmark of acute promyelocytic leukemia (APL) is a balanced reciprocal chromosomal translocation between chromosomes 15 and 17 leading to a fusion product consisting of the promyelocytic leukemia gene (PML) and the retinoic acid receptor alpha (RARA). The PML-RARA fusion product is necessary but not sufficient for the generation of leukemia and it is hypothesized that additional genomic lesions play a role in the pathogenesis of APL. Therefore, we have performed a high density SNP array analysis on 101 APL patient samples to identify new copy number alterations (CNAs) which may be relevant for the biology and prognosis of APL. Patients and Methods: Molecular and clinical outcome analyses were carried out retrospectively on patients diagnosed with APL, whose samples were referred to the molecular laboratory of the Department of Hematology and Oncology of the Medical Faculty Mannheim, University of Heidelberg, Germany between 1997 and 2010. 500 ng of genomic DNA from leukemic blasts per sample were processed according to the Genome Wide Human SNP 6.0 Array protocol (Affymetrix, Santa Clara, CA). The CNAG 3.3 software was used to perform allele-specific copy number analysis with anonymous references. CNAs of special interest were validated to be acquired in leukemia cells by performing allele-specific copy number analysis in matched pair SNP 6.0 array analysis, quantitative real time PCR and direct sequencing of genomic DNA from initial diagnosis and molecular remission samples. Results: We identified 279 acquired CNAs consisting of 185 heterozygous deletions, 87 amplifications and 7 regions of copy number neutral loss of heterozygosity (CNLOH). Besides common chromosomal aberrations such as trisomy 8 or duplications of the long arm of chromosome 8, deletions of 7q or isochromosome ider(17)(q10)t(15;17), numerous novel recurrent micro-deletions were discovered. The most common was a somatically acquired ∼100 kilobase deletion of chromosome 1q31.3 in 13 of 101 (13%) patients. These deletions encompassed the coding regions for the microRNAs mir181a1/b1. In univariate analyses of overall survival (OS) and relapse free survival (RFS) using Logrank tests, we found that patients carrying 2 or more CNAs as compared to 0 or 1 CNA as detected by SNP array had a significantly increased risk of death (p=0.016) and relapse (p=0.019). Patients carrying the recurrent deletion of chromosome 1q31.3 as compared to patients not carrying this deletion had a significantly increased risk of relapse (p=0.005), a markedly higher number of CNAs (median 8 vs. 2, p < 0.0001) and significantly higher white blood cell counts (WBC) at initial diagnosis (median 2550/μl vs. 16900/μl, p=0.009). We performed a multivariable analysis using Cox proportional hazards models to evaluate power of CNAs detected by SNP-array and deletions of chr. 1q31.3 as possible independent prognostic markers in APL as compared to age, WBC, platelet count (PC) and FLT3 mutational status. For the full model of OS only age and the number of CNAs detected by SNP-A met a 0.10 level of entry into the model and confirmed that the group of patients with 2 or more CNAs represent a subgroup with inferior outcome (“2 or more lesions”: hazard ratio = 5.942, p = 0.0015, age: hazard ratio = 1.08, p < 0.0001). Of note, for the analysis of RFS the presence of a chr. 1q31.3 deletion was the only effect, which met the entry level into the model of RFS and therefore was identified as a new strong predictor for an increased risk of relapse (“presence of del1q31.3”: hazard ratio = 28.9, p = 0.0031). Conclusion: The profiles of submicroscopic CNAs in APL patients are heterogeneic and may serve as strong independent prognostic markers for disease risk definition. Recurrent submicroscopic deletions of chr. 1q31.3 in leukemia cells of APL patients were associated with an increased number and characteristic pattern of further newly identified CNAs, unfavorable laboratory parameters and a higher risk of relapse. The number of CNAs was shown to be predictive for early death rate and overall survival. The further pursuit of these new potential molecular markers is highly warranted as they could refine the current risk stratification of APL by identifying new subgroups of patients, who could possibly gain from adapted treatment strategies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3543 Introduction: Acute promyelocytic leukemia (APL) is characterized by the translocation t(15;17) which results in the PML-RARα fusion protein. ATRA based treatment regimens lead to long term survival in approximately 75% of cases. However, relapse occurs in about 15% of APL patients. Moreover, it is not yet known which underlying pathomechanisms drive relapse of the disease. In addition, to avoid over- or undertreatment of patients, respectively, risk stratification of patients according to biological or molecular criteria would most probably improve current treatment modalities. As opposed to other leukemias, in APL no molecular markers have been established for risk stratification as yet. The gene brain and acute leukemia, cytoplasmic (BAALC) has been shown to be of prognostic relevance in other leukemias such as T acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Therefore, the aim of our study was to evaluate whether BAALC expression could be of prognostic value in APL. Materials and Methods: Bone marrow mononuclear cells were retrospectively evaluated in 86 APL patients after informed consent. 50 patients were female and 36 patients were male. Median age was 47 years (range: 19–82 years). All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) in triplicates on a LightCycler 480 (Roche, Mannheim, Germany). Glucose-6-phosphate-isomerase (GPI) served as a housekeeping gene for normalization. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used (Pfaffl MW, Nucleic Acids Res; 2001) after determination of PCR efficiencies. BAALC expression groups were defined as follows: BAALC expression lower than the 25th percentile (BAALClow) and higher than the 25th percentile (BAALC high). Time to complete remission, relapse free survival (RFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). For statistical analysis the Statistical Analysis Software version 9.2 was used. Results: In univariate analysis low BAALC expression was significantly associated with a superior OS. Patients in the BAALClow group showed an OS at 10 years of 82% as compared to 60% in the BAALChigh group (p=0.025). Moreover, RFS in the BAALClow group was significantly higher at 10 years as compared to the BAALChigh group (86% vs. 59%; p=0.0087). Time to complete remission was not correlated to BAALC expression levels. Conclusion: Low BAALC expression identifies a group of APL patients with a highly superior outcome indicated by a better OS and RFS in these patients. However, these findings need to be validated in an independent prospective study and it should be investigated whether this marker is independent of the applied treatment regimen and other known relevant markers. However, since the translocation t(15;17) by itself is not able to induce leukemia BAALC might be an interesting target for further investigation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1694 Introduction: In acute promyelocytic leukemia (APL) the t(15;17) is detectable in the vast majority of cases and leads to the fusion transcript PML-RARα. The introduction of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) into therapeutic management of APL resulted in long term remissions in about 80% of patients. However, relapse occurs in about 10–15% of cases and remains a major cause of treatment failure. The Wilms Tumor 1 gene (WT1) encodes for a transcription factor that is involved in normal and malignant hematopoiesis. Aberrant expression of WT1 has been shown to be associated with an adverse outcome in different types of hematological malignancies such as acute myeloid leukemia, acute lymphoblastic leukemia and myelodysplastic syndromes. To date only small series exist on aberrant WT1 expression in APL. Therefore, the aim of this study was to further elucidate the incidence of aberrant WT1 expression in APL and its prognostic significance for clinical endpoints. Methods: Bone marrow (BM) mononuclear cells were retrospectively evaluated in 89 APL patients (53 female, 36 male) and 21 healthy controls (15 female, 6 male) after informed consent. The median age of APL patients and healthy controls was 46 (range: 19–82 years) and 60 years (range: 21–84 years), respectively. All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) in triplicates on a LightCycler 480 (Roche, Mannheim, Germany). Beta–glucuronidase (GUS) served as a housekeeping gene for normalization. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used (Pfaffl MW, Nucleic Acids Res, 2001) after determination of PCR efficiencies. WT1 expression groups were defined as follows: WT1 expression higher than the 75th percentile (WT1high) and WT1 expression lower than the 25th percentile (WT1low). Time to complete remission, disease free survival (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). Results: Normalized WT1 expression was significantly higher in APL samples (median: 0.1129, range: 0.00001–1.414) as compared to normal controls (median: 0.00135, range: 0.00001–0.01105) (p=0.0011). WT1high was not significantly associated with high white blood cell counts (WBC). In univariate analysis time to complete remission was significantly shorter in patients showing high WT1 expression as compared to patients with low WT1 expression (47 days vs. 58 days, p=0.0051). However, this did not translate into a longer DFS or OS. In contrast, there was a trend toward an impaired DFS and OS in the WT1high group. Interestingly, analyzing deaths prior to complete remission WT1high was significantly associated with early death as compared to the WT1low group (median 6 days vs. 17 days, p=0.0197). Conclusion: WT1 is highly expressed in APL as compared to healthy controls but is not associated with increased WBC. WT1high was significantly associated with a shorter time to CR. Since WT1 plays a critical role in differentiation and survival in myeolopoiesis this finding might be due to a higher succeptibility of WT1high APL patients to ATRA induced differentiation as compared to the WT1low group. Moreover, in vitro studies have shown that in NB4 cells WT1 mRNA levels decrease upon treatment with ATRA, this provides further evidence that WT1 might be an additional target for ATRA in APL (Gu W, Haematologica, 2005). Interestingly, WT1high is significantly correlated with early death prior to CR achievement. We hypothezise that this might also be an effect of an increased toxicity of the ATRA based induction therapy in patients having high WT1 expression. Disclosures: Lengfelder: Cephalon: Research Funding.

Introduction: Recently we identified a recurrent acquired genomic deletion on chromosome 1q as a potential new marker in approximately 14% of APL patients predicting a significantly increased risk of relapse (Nowak D et al., Genes Chromosomes and Cancer 2012). The deleted region contains the coding sequences for the microRNAs hsa-mir-181a1 and hsa-mir-181b1, which have been implicated as prognostic factors in Acute Myeloid Leukemia (AML) and a corresponding host gene (MIR181A1HG). To elucidate biologic mechanisms associated with the described genomic deletion we performed targeted sequencing of the affected region and RNA sequencing of APL samples carrying the deletion versus samples not carrying the deletion with subsequent validation of novel variants of MIR181A1HG. Methods: Explorative sequencing of genomic DNA in the chromosomal subband 1q31.3, pos. 197073900-197196158 (hg18) was performed using the amplicon sequencing workflow of the Roche 454 platform sequencing 5000 bp fragments tiling a region of approximately 120 kb on n=3 APL samples. Corresponding patient samples from molecular remission were used as germline controls. Whole transcriptome sequencing of poly-A enriched RNA was performed on n=6 samples of bone marrow blasts of APL patients either carrying a deletion of the mir181a1/b1 coding region (n=3) or not carrying a deletion (n=3). RNA Sequencing was performed using the HiSeq2000 platform. Data analysis was carried out using Bowtie vers. 2.2.30, TopHat vers. 2.0.12 for alignment and mapping and the Cufflinks package vers. 2.2.1 for transcriptome assembly and expression analysis all using default settings and hg19 as reference genome. Validation of newly identified variants and differential expression of MIR181A1HG was carried out by RACE PCR and qRT-PCR on cDNA from primary leukemic blasts of APL patients (n=45), CD34+ cells from healthy donors (n=29). In vitro differentiation assays with concomitant gene expression analysis of MIR181A1HG variants were performed with CD34+ cells from healthy donors. Results: Genomic sequencing of the recurrently deleted region revealed no somatically acquired mutations in the analyzed APL samples. Differential gene expression analysis using FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) inferred from RNA sequencing data of APL samples carrying a genomic deletion of 1q31.3 versus non-deleted samples identified n=58 genes significantly downregulated in deleted samples and n=31 upregulated genes. Interestingly, among the differentially regulated genes, BAALC, a factor recently shown to be prognostically relevant in APL was significantly upregulated 13 fold in the unfavourable group of samples with 1q31.3 deletions. Furthermore, RNA sequencing revealed numerous new isoforms of known transcripts as well as novel long non-conding RNA (lncRNA) sequences. Among these were a total of 6 new transcript variants of the MIR181A1HG gene in the recurrently deleted region on chromosome 1q31.3. One novel 5600bp lncRNA covering the coding regions for the hsa-mir-181a1/b1 was 24 fold overexpressed in samples carrying the recurrent 1q31.3 deletions. Expression analysis of MIR181A1HG in blasts of APL patients, CD34+ cells, unselected bone marrow cells and granulocytes of healthy donors revealed significantly elevated levels of MIR181A1HG in APL cells as compared to healthy CD34+ cells and almost absent expression in unselected bone marrow and granulocytes. This indicated a possible role for MIR181A1HG in APL blasts and hematopoietic stem cells. Subsequent in vitro differentiation experiments of primary healthy CD34+ cells showed that MIR181A1HG is downregulated 7 fold within 14 days of cytokine induced myeloid differentiation. Furthermore, MIR181A1HG was downregulated 5 fold during ATRA induced differentiation of NB4 cells. Conclusion: RNA sequencing of APL cells demonstrated numerous novel uncharacterized lncRNAs whose expression is associated with clinical risk and which merit further investigation. Identification of novel isoforms of MIR181A1HG, which are highly expressed in APL blasts and purified CD34+ cells suggest a potential role for this lncRNA in hematopoietic stem cells and response to ATRA induced differentiation of APL cells. Disclosures No relevant conflicts of interest to declare.