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Growth/differentiation factor−15 (Gdf-15) is a member of the transforming growth factor-β (Tgf-β) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson’s disease (PD) by comparing Gdf-15 +/+ and Gdf-15 −/− mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 +/+ and Gdf-15 −/− mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 +/+ and Gdf-15 −/− mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

The combination of 5-azacytidine (AZA) with donor lymphocyte infusions (DLIs) can induce remissions in patients with relapsed myeloid malignancies after allo-HCT. As decitabine (DAC) is known to be effective also in AML/MDS with leukocytosis, we investigated the combination of DAC with DLIs for relapse after allo-HCT. Between 2006 and 2016, 26 patients (median age 59 years) with AML (n = 18), MDS (n = 6), or MPN (n = 2) and overt hematological relapse after allo-HCT were treated. Median duration from allo-HCT to relapse was 306 days (range, 76-4943). Eighteen patients received DAC + DLIs, 8 DAC-only (median number cycles of DAC: 2, range 1-13, median number of DLIs: 2, range 1-10). The incidence of acute and chronic GvHD in patients receiving DLI was 17% (3/18) and 6% (1/18), respectively. CR/CRi was achieved in 15% (4/26), PR in 4% (1/26), and stable disease in 58% (15/26) of patients. Eight patients received a second allo-HCT. Median overall survival was 4.7 months. Elevated PD-L1 protein expression in bone marrow cells was detected in 4/8 patients with > 20% blast infiltration prior to DAC, without a clear association with response. In conclusion, the DAC + DLI regimen proved feasible and effective in relapsed myeloid malignancies after allo-HCT, with efficacy not restricted to patients with low leukemic burden.

Experimental model systems identified phosphorylation of linker histone variant H1.4 at Ser 27 (H1.4S27p) as a novel mitotic mark set by Aurora B kinase. Here, we examined expression of Aurora B and H1.4S27p in colorectal carcinoma (CRC) cell lines (HCT116, DLD1, Caco-2, HT29) and tissue specimens (n = 36), in relation to microsatellite instability (MSI) status and ploidy. In vitro, Aurora B (pro-/meta-/anaphase) and H1.4S27p (pro-/metaphase) were localized in mitotic figures. The proportion of labeled mitoses was significantly different between cell lines for Aurora B (p = 0.019) but not for H1.4S27p (p = 0.879). For Aurora B, these differences were not associated with an altered Aurora B gene copy number (FISH) or messenger RNA (mRNA) expression level (qRT-PCR). Moreover, Aurora B expression and H1.4S27 phosphorylation were no longer coordinated during metaphase in aneuploid HT29 cells (p = 0.039). In CRCs, immunoreactivity for Aurora B or H1.4S27p did not correlate with T- or N-stage, grade, or MSI status. However, metaphase labeling of H1.4S27p was significantly higher in diploid than in aneuploid CRCs (p = 0.011). Aurora B was significantly correlated with H1.4S27p-positive metaphases in MSI (p = 0.010) or diploid (p = 0.003) CRCs. Finally, combined classification of MSI status and ploidy revealed a significant positive correlation of Aurora B with H1.4S27p in metaphases of diploid/MSI (p = 0.010) and diploid/microsatellite-stable (MSS; p = 0.031) but not of aneuploid/MSS (p = 0.458) CRCs. The present study underlines the functional link of Aurora B expression and H1.4S27p during specific phases of mitosis in diploid and/or MSI-positive CRCs in vitro and in situ. Importantly, the study shows that the coordination between Aurora B expression and phosphorylation of H1.4 at Ser 27 is lost in cycling aneuploid CRC cells.

Improving mid-term and long-term outcomes after solid organ transplantation is imperative, and requires both state-of-the-art transplant surgery and optimization of routine, evidence-based aftercare. This randomized, controlled trial assessed the effectiveness of standard aftercare versus telemedically supported case management, an innovative aftercare model, in 46 living-donor renal transplant recipients during the first posttransplant year. The model includes three components: (i) chronic care case management initiated after discharge, (ii) case management initiated in emerging acute care situations, and (iii) a telemedically equipped team comprising a transplant nurse case manager and two senior transplant physicians (nephrologist, surgeon). Analyses revealed a reduction of unplanned inpatient acute care, with considerable cost reductions, in the intervention group. The prevalence of nonadherence over the 1-year study period was 17.4% in the intervention group versus 56.5% in the standard aftercare group (p = 0.013). Only the intervention group achieved their pre-agreed levels of adherence, disease-specific quality of life, and return to employment. This comparative effectiveness study provides the basis for multicenter study testing of telemedically supported case management with the aim of optimizing posttransplant aftercare. The trial was registered with the German Clinical Trials Register (www.DRKS.de), DKRS00007634. ispartof: American Journal of Transplantation vol:17 issue:6 pages:1594-1605 ispartof: location:United States status: published

The inhibitors of DNA binding (ID) inhibit basic helix-loop-helix transcription factors and thereby guide cellular differentiation and proliferation. To elucidate the involvement of IDs in hematopoiesis and acute leukemias (AL), we analyzed ID2 and ID3 expression in hematopoiesis and leukemic blasts in bone marrow biopsies (BMB). BMB of healthy stem cell donors (n = 19) and BMB of patients with acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MD; n = 19), de novo AML (n = 20), B-acute lymphoblastic leukemia (B-ALL) (n = 23), T-ALL (n = 19), were immunohistochemically stained for ID2 and ID3 expression. The expression patterns were evaluated and quantified for each hematopoietic lineage and each leukemia subtype. In normal BMB, immature granulopoiesis showed weak ID2 and strong ID3 expression, which was lost during maturation (p < 0.001). Erythropoiesis remained negative for ID2/3 (p < 0.001). ID2/3 expression differed between immature granulopoiesis and leukemic blasts (p < 0.001). Moreover, differential ID2/3 expression was seen between AL subgroups: AML, especially AML-MD, had more ID2- (p < 0.001) and ID3-positive (p < 0.001) blasts than ALL. We show a comprehensive in situ picture of ID2/3 expression in hematopoiesis and AL. Morphologically, ID2/3 proteins seem to be involved in the granulopoietic maturation. Importantly, the distinct ID2/3 expression patterns in AL indicate a specific deregulation of ID2/3 in the various types of AL and may support subtyping of AL.

Postoperative pulmonary and renal complications are frequent in patients undergoing lung surgery. Hyper- and hypovolaemia may contribute to these complications. We hypothesized that goal-directed haemodynamic management based on oesophageal Doppler monitoring would reduce postoperative pulmonary complications in a randomized clinical parallel-arm trial.One hundred patients scheduled for thoracic surgery were randomly assigned to either standard haemodynamic management (control group) or goal-directed therapy (GDT group) guided by an oesophageal Doppler monitoring-based algorithm. The primary endpoint was postoperative pulmonary complications, including spirometry. Secondary endpoints included haemodynamic variables, renal, cardiac, and neurological complications, and length of hospital stay. The investigator assessing outcomes was blinded to group assignment.Forty-eight subjects of each group were analysed. Compared to the control group, fewer subjects in the GDT group developed postoperative pulmonary complications (6 vs. 15 patients; P = 0.047), while spirometry did not differ between groups. Compared to the control group, patients of the GDT group showed higher cardiac index (2.9 vs. 2.1 [l min - 1 m - 2 ]; P 0.001) and stroke volume index (43 vs. 34 [ml m 2 ]; P 0.001) during surgery. Renal, cardiac and neurological complications did not differ between groups. Length of hospital stay was shorter in the GDT compared to the control group (9 vs. 11 days; P = 0.005).Compared to standard haemodynamic management, oesophageal Doppler monitor-guided GDT was associated with fewer postoperative pulmonary complications and a shorter hospital stay.The study was registered in the German Clinical Trials Register (DRKS 00006961). https://drks-neu.uniklinik-freiburg.de/drks_web/.

// Marta Michalska 1 , Susanne Schultze-Seemann 1 , Lioudmila Bogatyreva 2 , Dieter Hauschke 2 , Ulrich Wetterauer 1 , Philipp Wolf 1 1 Department of Urology, Medical Center, University of Freiburg, Freiburg, Germany 2 Institute for Medical Biometry and Statistics, Medical Center, University of Freiburg, Freiburg, Germany Correspondence to: Philipp Wolf, e-mail: philipp.wolf@uniklinik-freiburg.de Keywords: prostate cancer, PSMA, immunotoxin, docetaxel, chemotherapy Received: November 26, 2015 Accepted: February 23, 2016 Published: March 9, 2016 ABSTRACT Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments. In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo . The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC 50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC.

DNA-hypomethylating agents are a viable treatment option for AML/myelodysplastic syndrome (MDS) relapse after allograft by upregulating Ags on blasts before DLI. Seventy-two patients with relapsed AML (n=62), MDS (n=8) and other myeloid neoplasms (n=2) after allograft were treated with low-dose 5-azacytidine and, if feasible, DLI. Patient characteristics: median age 62 years (range 20–75), 42% with adverse cytogenetics, 82% not in remission at transplant and 83% received fludarabine-based reduced-toxicity conditioning. Median duration from transplant to 5-azacytidine was 289 days (range 59–2133). Response criteria: CR, temporary disease control or treatment failure. A median of 2.7 courses (range 1–10) were administered; 65 out of 72 patients also received DLI (41 already before 5-azacytidine). Ten patients developed acute GVHD and two succumbed to treatment-related sepsis. CR rate was 9.7% (in two patients lasting >5 years), 44% had temporary disease control (median duration 71 days, range 31–380). Median survival from 5-azacytidine was 108 days, 21 patients proceeded to subsequent transplant. In multivariate analysis, peripheral blood blasts

Polo-like kinase 1 (PLK1) is an important regulator of the cell cycle and is overexpressed in various solid and hematological malignancies. Small molecule inhibitors targeting PLK1, such as BI2536 or BI6727 (Volasertib) are a promising therapeutic approach in such malignancies. Here, we show a loss of specifically localized PLK1 in AML blasts in vivo, accompanied by mitotic arrest with transition into apoptosis, in bone marrow biopsies of AML patients after treatment with BI2536. We verify these results in live cell imaging experiments with the AML cell line HL-60, and demonstrate that non-neoplastic, immortalized lymphoblastoid cells are also sensitive to PLK1 inhibition. It is demonstrated that normal granulopoietic precursors have similar PLK1 expression levels as leukemic blasts. These results are in line with the adverse effects of PLK1 inhibition and underline the great potential of PLK1 inhibitors in the treatment of AML.

Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC, n=49) and Barrett's adenocarcinomas (BAC, n=61) revealed similar STAT3 expression in ESCCs and BACs (P=0.109), but preferentially activated P-STAT3 in ESCCs (P=0.013). In vitro, strong STAT3 activation was seen by epidermal growth factor (EGF) stimulation in OE21 (ESCC) cells, whereas OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 (P=0.0148) and OE33 (P=0.0243) cells. Importantly, STAT3 knockdown reduced cell migration of OE33 cells by 2.5-fold in two types of migration assays (P=0.073, P=0.015), but not in OE21 cells (P=0.1079, P=0.386). Investigation of transcriptome analysis of STAT3 knockdown revealed a reduced STAT3 level associated with significant downregulation of cell cycle genes in both OE21 (P

Aims Aurora kinases are central to cell proliferation and considered to be prognostic/predictive markers and therapeutic targets for epithelial cancers. Here, the prognostic/predictive value of Aurora-B protein expression was evaluated in patients with serous, FIGO stage III ovarian carcinomas treated with taxane- or platinum-based first-line chemotherapy (1st-CTx). Methods Immunohistochemistry was performed on tissue microarrays, including 80 ovarian carcinomas and 18 non-neoplastic ovaries, previously characterised for Aurora-A protein expression. None or marginal (score 0+1), moderate (score 2) and strong (score 3) Aurora-B protein expression was correlated with clinico-pathological parameters as well as recurrence-free survival (RFS) and overall survival (OS). Results While non-neoplastic ovaries were negative for Aurora-B, almost all (79/80; 99%) ovarian carcinomas exhibited Aurora-B positive tumour cells, with score 1 in 41/80 (51%), score 2 in 23/80 (29%) and score 3 in 15/80 (19%) cases. Aurora-B and Aurora-A protein expression correlated significantly (p=0.002). In optimal debulked patients, Aurora-B protein expression was associated with RFS (p=0.011, n=53) and marginally with OS (p=0.460; n=53). Moreover, Aurora-B protein expression was predictive for RFS of optimal debulked patients with taxane-based (p=0.006; n=32), but not with platinum-based (p=0.720; n=20) 1st-CTx. Aurora-B protein expression was not linked to OS in optimal debulked patients with either of the two 1st-CTx. Conclusions Aurora-B protein expression frequently occurs in serous, FIGO stage III ovarian carcinomas, making it a ‘drugable’ molecular target in the majority of ovarian carcinoma patients. Moreover, Aurora-B protein expression is predictive for initial response to taxane-based 1st-CTx in optimal debulked, late stage ovarian carcinoma patients.

Introduction: Aberrant DNA methylation is frequently found in hematologic malignancies where it is associated with altered gene expression. DNA hypomethylating agents (DNMTi), e.g. 5-aza-2'-deoxycytidine (DAC), are used for both global and gene-specific in vivo demethylation and offer a therapeutic option in myelodysplastic syndromes (MDS) and AML. DNMTi have already been utilized to upregulate suppressed fetal hemoglobin (HbF) in adult patients (pts) suffering from hemoglobinopathies. Here we systematically investigated the potential of DAC for in vitro induction of erythroid differentiation as well as HbF expression in the bipotent myeloid leukemia cell line K562 and in vivo in a clinical treatment situation in MDS pts. Methods and Results: We treated K562 cells with non-toxic concentrations of DAC (100 nM, three 24 hour pulses), hemin (50 nM) and phorbol myristate acetate (PMA, 5 nM). DAC treatment led to morphological changes indicating erythroid but not megakaryocytic differentiation. This was confirmed by benzidine staining where DAC (13% positive cells) and hemin (58%) but not PMA treated cells (0%) became positive for hemoglobin synthesis. Lack of CD41 detection by FACS analysis for DAC and hemin indicated absence of megakaryocytic differentiation. Transcriptome profiling by mRNA expression arrays (Affymetrix GeneChip® HG U133 Plus 2.0) revealed highest similarity between hemin and DAC treatment by unsupervised hierarchical clustering, followed by vehicle control and untreated cells. The transcriptome of PMA treated cells clustered most distantly to all other treatments. Both, DAC and hemin induced moderately balanced up- and downregulation of transcripts to an almost identical extent. 1414 transcripts were >2 fold upregulated and 1505 were >2 fold downregulated upon DAC treatment, whereas 1548 were up- and 2404 were downregulated in hemin treated cells, respectively. The extent of transcriptome dynamics was considerably stronger upon PMA treatment, where 4196 and 3780 transcripts were up- and downregulated, respectively. When intersecting transcriptome changes between the 3 drug treatments (Fig. 1), 368 out of 1548 (23.7%) upregulated transcripts in hemin treated cells were concordantly upregulated upon DAC treatment. The overlap of upregulated transcript was lower compared to PMA treated cells (14.9%). GO analyses of upregulated transcripts identified terms related to erythropoesis and iron metabolism among the top regulated groups of transcripts in DAC treated cells whereas terms related to megakaryocytic differentiation did not show significance. Particularly strong differences of transcripts were observed for a1-, a2-, Ag-, e- and z-globin expression upon DAC and hemin treatment, whereas b- and d-globin were expressed at low levels. These changes were not observed for PMA treated cells. Induction of a- and Ag-globin on mRNA level resulted in enrichment of a- and Ag-globin protein to 15.8% of total cellular protein amount, and consequently in HbF formation in K562 cells as assessed by reversed phase and anion exchange chromatography. HbF levels in peripheral blood were measured from 16 MDS pts, median age 74 years (range 66-78) also treated with a 3-day DAC schedule. Median HbF fraction at baseline was 0.4% (0.1-3.9%) of total hemoglobin with 6 pts (37.5%) exhibiting increased HbF levels (>1%) already before treatment. In 13/16 (81%) pts, increase of HbF with a median increment of 1.2% (range 0.3-3.7%) was observed. In 3 pts, HbF decreased over the treatment course. Median number of courses until maximum increment was 3 (2-6). HbF levels in 2 pts with AML and 1 with pancreatic cancer treated with nucleoside analogues without demethylating activity (cytosine arabinoside and gemcitabine, respectively) according to standard chemotherapy protocols served as control group and did not show comparable increments. Conclusions: We describe an erythroid differentiation program, from transcriptome level to HbF protein formation, induced by the hypomethylating agent DAC in the bipotent cell line K562. This DAC-mediated differentiation process is specific for erythropoesis but not megakaryopoesis. This is substantiated by in vivo upregulation of HbF upon DAC adminstration in MDS pts. Therefore, we propose to utilize HbF expression as potential biomarker during DAC treatment. Figure 1. Intersection of >2 fold upregulated transcripts in K562 cells upon drug treatment. Figure 1. Intersection of >2 fold upregulated transcripts in K562 cells upon drug treatment. Disclosures No relevant conflicts of interest to declare.

Background The DNA hypomethylating agents 5-azacytidine (aza) and decitabine are a new treatment option for AML/MDS pts relapsing after allografting. Cancer/testis antigens are upregulated in vitro and in vivo by these drugs (Goodyear et al., Blood 2010, Almstedt et al., Leuk. Res. 2010), hypothetically sensitizing (“priming”) malignant cells to DLIs. In 2006 we initiated a program of administering aza (100 mg/day) for 3 consecutive days, followed whenever possible by DLI 7 days after the last aza injection (repeated every 3-4 weeks). Of 26 pts treated thus, more than 50% achieved at least temporary disease control, with 4 pts achieving complete remissions (CRs) of >12 months duration at time of reporting (Lübbert et al., BMT 2009). We now updated the results of these 26 pts and report on the further 46 pts treated on this program. Patients and Methods Between 10/2006 and 05/2013, 72 pts (median age 62 years, range 20–75, 46 male, 26 female) with hematological relapse after allografting were treated with aza and, if at all feasible, with DLI as described above. A 5-day schedule of 100 mg/m2 aza daily as starting dose was introduced in 2009 for pts with higher leukemic load. Diagnoses were AML (n=62), MDS (n=6), CMMoL (n=2), acute mast cell leukemia and blastic plasmacytoid dendritic cell neoplasm (1 each). Of 68 pts with informative cytogenetics, 44 had intermediate-2 or adverse cytogenetics according to ELN Guidelines. Sixty pts had received fludarabine-based reduced-toxicity conditioning regimens (mostly FBM), 12 had received myeloablative conditioning; 23 pts had a HLA-matched sibling donor, 47 pts an unrelated donor and 2 pts a syngeneic sibling donor. Median duration from transplant to first day of aza was 289 days (range, 59 to 2133), 48,19 and 2 pts were in 1st, 2nd or 3rd hematologic relapse, respectively, at start of aza. Two pts had only mixed chimerism, one had only extramedullary disease at 1st relapse. Median % of blasts before aza was 4 % (range 0 to 91) in peripheral blood and 25 % (range 0 to 100) in bone marrow. Response criteria (for CR, temporary disease control or treatment failure) were applied as previously described. Uni- and multivariate analyses were performed. Results 47 pts (65 %) and 18 pts (25 %) received a 3-day or 5-day course of aza, respectively, as starting dose. In 3 (4 %) and 4 pts (6 %), initial dosing was limited to 2 and 4 days, respectively. A total of 189 courses of aza were administered (median of 2.7 courses, range 1 to 10). 65/72 pts (90 %) also received DLIs, 41/72 (57%) already prior to first aza. Only 7 pts developed acute GvHD after DLI treatment. Clinically relevant neutropenic infections not associated with disease progression developed in 4 pts, in one of them leading to fatal sepsis. 39 pts (54 %) benefited from the treatment, with CR in 7 pts (9.7%), in 2 of them now lasting more than 5 yrs. Thirty-two pts (44 %) had temporary disease control with stable mixed chimerism (median duration 71 days, range 31-380), with 2 pts converting to complete donor chimerism lasting 146 and 380 days, respectively. The median survival from start of aza was 108 days. Nine pts are alive at the time of this report. Twenty-one pts proceeded to subsequent transplant, 4 of them (19%) are presently alive. Among 10 predictive factors, 5 were significant at p Conclusion In an extended cohort of pts we could confirm our previous results of clinical activity of this approach. Notably, there are long-term remitters after this well-tolerated outpatient treatment, which appears particularly effective in pts without peripheral blood blasts. Recurrent responses to the aza / DLI combination in pts who had progressed under DLI-only treatment support the “priming” concept whereby hypomethylating agents can sensitize malignant cells to other biological or cellular therapies, thus acting as so-called biological response modifiers. Disclosures: Off Label Use: 5-azacytidine, immunomodulation.