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In newly diagnosed acute myeloid leukemia, immediate initiation of treatment is standard of care. However, deferral of antileukemic therapy may be indicated to assess comorbidities or pre-therapeutic risk factors. We explored the impact of time from diagnosis to treatment on outcomes in newly diagnosed acute myeloid leukemia undergoing venetoclax-based therapy in two distinct cohorts. By querying the Study Alliance Leukemia database and the global health network TriNetX, we identified 138 and 717 patients respectively with an average age of 76 and 72 years who received venetoclax-based firstline therapy. When comparing patients who started treatment earlier or later than 10 days after initial diagnosis, no significant difference in median overall survival was observed - neither in the SAL cohort (7.7 vs. 9.6 months, p=.42) nor in the TriNetX cohort (7.5 vs. 7.2 months, p=.41). Similarly, severe infections, bleeding, and thromboembolic events were equally observed between early and later treatments, both in the overall patient groups and specific subgroups (age ≥75 years or leukocytes ≥20x109/L). This retrospective analysis indicates that delaying the start of venetoclax-based therapy in newly diagnosed acute myeloid leukemia might be a safe option for selected patients, provided that close clinical monitoring is performed.

Abstract Tandem-duplication mutations of the UBTF gene (UBTF-TDs) coding for the upstream binding transcription factor have recently been described in pediatric patients with acute myeloid leukemia (AML) and were found to be associated with particular genetics (trisomy 8 (+8), FLT3-internal tandem duplications (FLT3-ITD), WT1-mutations) and inferior outcome. Due to limited knowledge on UBTF-TDs in adult AML, we screened 4247 newly diagnosed adult AML and higher-risk myelodysplastic syndrome (MDS) patients using high-resolution fragment analysis. UBTF-TDs were overall rare (n = 52/4247; 1.2%), but significantly enriched in younger patients (median age 41 years) and associated with MDS-related morphology as well as significantly lower hemoglobin and platelet levels. Patients with UBTF-TDs had significantly higher rates of +8 (34% vs. 9%), WT1 (52% vs. 7%) and FLT3-ITD (50% vs. 20.8%) co-mutations, whereas UBTF-TDs were mutually exclusive with several class-defining lesions such as mutant NPM1, in-frame CEBPA bZIP mutations as well as t(8;21). Based on the high-variant allele frequency found and the fact that all relapsed patients analyzed (n = 5) retained the UBTF-TD mutation, UBTF-TDs represent early clonal events and are stable over the disease course. In univariate analysis, UBTF-TDs did not represent a significant factor for overall or relapse-free survival in the entire cohort. However, in patients under 50 years of age, who represent the majority of UBTF-mutant patients, UBTF-TDs were an independent prognostic factor for inferior event-free (EFS), relapse-free (RFS) and overall survival (OS), which was confirmed by multivariable analyses including established risk factors such as age and ELN2022 genetic risk groups (EFS [HR: 2.20; 95% CI 1.52–3.17, p

Abstract Background The role of allogeneic hematopoietic cell transplantation (alloHCT) in acute myeloid leukemia (AML) with mutated IDH1/2 has not been defined. Therefore, we analyzed a large cohort of 3234 AML patients in first complete remission (CR1) undergoing alloHCT or conventional chemo-consolidation and investigated outcome in respect to IDH1/2 mutational subgroups (IDH1 R132C, R132H and IDH2 R140Q, R172K). Methods Genomic DNA was extracted from bone marrow or peripheral blood samples at diagnosis and analyzed for IDH mutations with denaturing high-performance liquid chromatography, Sanger sequencing and targeted myeloid panel next-generation sequencing, respectively. Statistical as-treated analyses were performed using R and standard statistical methods (Kruskal–Wallis test for continuous variables, Chi-square test for categorical variables, Cox regression for univariate and multivariable models), incorporating alloHCT as a time-dependent covariate. Results Among 3234 patients achieving CR1, 7.8% harbored IDH1 mutations (36% R132C and 47% R132H) and 10.9% carried IDH2 mutations (77% R140Q and 19% R172K). 852 patients underwent alloHCT in CR1. Within the alloHCT group, 6.2% had an IDH1 mutation (43.4% R132C and 41.4% R132H) and 10% were characterized by an IDH2 mutation (71.8% R140Q and 24.7% R172K). Variants IDH1 R132C and IDH2 R172K showed a significant benefit from alloHCT for OS (p = .017 and p = .049) and RFS (HR = 0.42, p = .048 and p = .009) compared with chemotherapy only. AlloHCT in IDH2 R140Q mutated AML resulted in longer RFS (HR = 0.4, p = .002). Conclusion In this large as-treated analysis, we showed that alloHCT is able to overcome the negative prognostic impact of certain IDH mutational subclasses in first-line consolidation treatment and could pending prognostic validation, provide prognostic value for AML risk stratification and therapeutic decision making.

Abstract Background Extramedullary manifestations (EM) are rare in acute myeloid leukemia (AML) and their impact on clinical outcomes is controversially discussed. Methods We retrospectively analyzed a large multi-center cohort of 1583 newly diagnosed AML patients, of whom 225 (14.21%) had EM. Results AML patients with EM presented with significantly higher counts of white blood cells (p

FLT3-ITD mutations are common druggable alterations in patients with acute myeloid leukemia (AML) and associated with poor prognosis. Beside typical ITD mutations, point mutations and deletions in the juxtamembrane domain (JMD) have been observed. However, due to the low frequency of these alterations, there is only limited information on molecular and clinical associations. To evaluate the prognostic impact of non-ITD mutations in the FLT3 JMD region, we analyzed a large cohort of 1,539 adult AML patients treated in different protocols of the Study Alliance Leukemia, using next-generation sequencing. Non-ITD point mutations and deletions within the FLT3 JMD were identified with a prevalence of ~1.23% (n = 19). Both FLT3-ITD and non-ITD mutations were associated with a higher rate of NPM1 (42%–61%; p < 0.001) and DNMT3A mutations (37%–43%; p < 0.001), as well as an increased percentage of peripheral blood (54%–65%) and bone marrow blast cells (74%; p < 0.001), compared to FLT3-wild-type patients. Most significantly, AML patients with FLT3 non-ITD mutations had a higher rate of concomitant KMT2A-PTD mutations (37.5%; p < 0.001) as compared to FLT3-ITD (7%) or FLT3-wild-type cases (4.5%). In a multivariable analysis, FLT3 non-ITD mutations were not an independent prognostic factor. However, patients with dual FLT3 non-ITD and KMT2A-PTD mutations showed a trend for inferior outcome, which points at a functional interaction in this subset of AML.

Chimeric antigen receptor T cells (CAR-T) targeting CD19 or B cell maturation antigen (BCMA) are highly effective against B cell malignancies. However, application of CAR-T to less differentially expressed targets remains a challenge due to lack of tumor-specific antigens and CAR-T controllability. CD123, a highly promising leukemia target, is expressed not only by leukemic and leukemia-initiating cells, but also by myeloid, hematopoietic progenitor, and certain endothelial cells. Thus, CAR-T lacking fine-tuned control mechanisms pose a high toxicity risk. To extend the CAR-T target landscape and widen the therapeutic window, we adapted our rapidly switchable universal CAR-T platform (UniCAR) to target CD123. UniCAR-T efficiently eradicated CD123+ leukemia in vitro and in vivo. Activation, cytolytic response, and cytokine release were strictly dependent on the presence of the CD123-specific targeting module (TM123) with comparable efficacy to CD123-specific CAR-T in vitro. We further demonstrated a pre-clinical proof of concept for the safety-switch mechanism using a hematotoxicity mouse model wherein TM123-redirected UniCAR-T showed reversible toxicity toward hematopoietic cells compared to CD123 CAR-T. In conclusion, UniCAR-T maintain full anti-leukemic efficacy, while ensuring rapid controllability to improve safety and versatility of CD123-directed immunotherapy. The safety and efficacy of UniCAR-T in combination with TM123 will now be assessed in a phase I clinical trial (ClinicalTrials.gov: NCT04230265).

The detection of plasma cell–free tumor DNA (ctDNA) is prognostic in colorectal cancer (CRC) and has potential for early prediction of disease recurrence. In clinical routine, ctDNA-based diagnostics are limited by the low concentration of ctDNA and error rates of standard next-generation sequencing (NGS) approaches. We evaluated the potential to increase the stability and yield of plasma cell–free DNA (cfDNA) for routine diagnostic purposes using different blood collection tubes and various manual or automated cfDNA extraction protocols. Sensitivity for low-level ctDNA was measured in KRAS-mutant cfDNA using an error-reduced NGS procedure. To test the applicability of rapid evaluation of ctDNA persistence in clinical routine, we prospectively analyzed postoperative samples of 67 CRC (stage II) patients. ctDNA detection was linear between 0.0045 and 45%, with high sensitivity (94%) and specificity (100%) for mutations at 0.1% VAF. The stability and yield of cfDNA were superior when using Streck BCT tubes and a protocol by Zymo Research. Sensitivity for ctDNA increased 1.5-fold by the integration of variant reads from triplicate PCRs and with PCR template concentration. In clinical samples, ctDNA persistence was found in ∼9% of samples, drawn 2 weeks after surgery. Moreover, in a retrospective analysis of 14 CRC patients with relapse during adjuvant therapy, we successfully detected ctDNA (median 0.38% VAF; range 0.18–5.04% VAF) in 92.85% of patients significantly prior (median 112 days) to imaging-based surveillance. Using optimized pre-analytical conditions, the detection of postoperative ctDNA is feasible with excellent sensitivity and allows the prediction of CRC recurrence in routine oncology testing.

Chimeric antigen receptor T cells (CAR-T) targeting CD19 have achieved significant success in patients with B cell malignancies. To date, implementation of CAR-T in other indications remains challenging due to the lack of truly tumor-specific antigens as well as control of CAR-T activity in patients. CD123 is highly expressed in acute myeloid leukemia (AML) blasts including leukemia-initiating cells making it an attractive immunotherapeutic target. However, CD123 expression in normal hematopoietic progenitor cells and endothelia bears the risk of severe toxicities and may limit CAR-T applications lacking fine-tuned control mechanisms. Therefore, we recently developed a rapidly switchable universal CAR-T platform (UniCAR), in which CAR-T activity depends on the presence of a soluble adapter called targeting module (TM), and confirmed clinical proof-of-concept for targeting CD123 in AML with improved safety. As costimulation via 4–1BB ligand (4–1BBL) can enhance CAR-T expansion, persistence, and effector functions, a novel CD123-specific TM variant (TM123-4-1BBL) comprising trimeric single-chain 4–1BBL was developed for transient costimulation of UniCAR-T cells (UniCAR-T) at the leukemic site in trans. TM123-4-1BBL-directed UniCAR-T efficiently eradicated CD123-positive AML cells in vitro and in a CDX in vivo model. Moreover, additional costimulation via TM123-4-1BBL enabled enhanced expansion and persistence with a modulated UniCAR-T phenotype. In addition, the increased hydrodynamic volume of TM123-4-1BBL prolonged terminal plasma half-life and ensured a high total drug exposure in vivo. In conclusion, expanding the soluble adapter optionality for CD123-directed UniCAR-T maintains the platforms high anti-leukemic efficacy and immediate control mechanism for a flexible, safe, and individualized CAR-T therapy of AML patients.

Extramedullary (EM) disease in patients with acute myeloid leukemia (AML) is a known phenomenon. Since the prevalence of EM AML has so far only been clinically determined on examination, we performed a prospective study in patients with AML. The aim of the study was to determine the prevalence of metabolically active EM AML using total body 18Fluorodesoxy-glucose positron emission tomography/computed tomography (18FDG-PET/CT) imaging at diagnosis prior to initiation of therapy. In order to define the dynamics of EM AML throughout treatment, PET-positive patients underwent a second 18FDG-PET/CT imaging series during follow up by the time of remission assessment. A total of 93 patients with AML underwent 18FDG-PET/CT scans at diagnosis. The prevalence of PET-positive EM AML was 19% with a total of 65 EM AML manifestations and a median number of two EM manifestations per patient (range, 1-12), with a median maximum standardized uptake value of 6.1 (range, 2-51.4). When adding those three patients with histologically confirmed EM AML who were 18FDG-PET/CT negative in the 18FDG-PET/CT at diagnosis, the combined prevalence for EM AML was 22%, resulting in 77% sensitivity and 97% specificity. Importantly, 60% (6 of 10) patients with histologically confirmed EM AML still had active EM disease in their follow up 18FDG-PET/CT. 18FDG-PET/CT reveals a high prevalence of metabolically active EM disease in AML patients. Metabolic activity in EM AML may persist even beyond the time point of hematologic remission, a finding that merits further prospective investigation to explore its prognostic relevance. (Trial registered at clinicaltrials.gov identifier: 01278069.)

Acute myeloid leukemia (AML) with t(6;9)(p22;q34) is a distinct entity accounting for 1-2% of AML cases. A substantial proportion of these patients have a concomitant FLT3-ITD. While outcomes are dismal with intensive chemotherapy, limited evidence suggests allogeneic hematopoietic cell transplantation (allo-HCT) may improve survival if performed early during first complete remission. We report on a cohort of 178 patients with t(6;9)(p22;q34) within an international, multicenter collaboration. Median age was 46 years (range: 16-76), AML was de novo in 88%, FLT3-ITD was present in 62%, and additional cytogenetic abnormalities in 21%. Complete remission was achieved in 81% (n=144), including 14 patients who received high-dose cytarabine after initial induction failure. With a median follow up of 5.43 years, estimated overall survival at five years was 38% (95%CI: 31-47%). Allo-HCT was performed in 117 (66%) patients, including 89 in first complete remission. Allo-HCT in first complete remission was associated with higher 5-year relapse-free and overall survival as compared to consolidation chemotherapy: 45% (95%CI: 35-59%) and 53% (95%CI: 42-66%) versus 7% (95%CI: 3-19%) and 23% (95%CI: 13-38%), respectively. For patients undergoing allo-HCT, there was no difference in overall survival rates at five years according to whether it was performed in first [53% (95%CI: 42-66%)], or second [58% (95%CI: 31-100%); n=10] complete remission or with active disease/relapse [54% (95%CI: 34-84%); n=18] (P=0.67). Neither FLT3-ITD nor additional chromosomal abnormalities impacted survival. In conclusion, outcomes of t(6;9)(p22;q34) AML are poor with chemotherapy, and can be substantially improved with allo-HCT.

Abstract: Patients with secondary AML (sAML) with antecedent myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPNs) tend to have high-risk disease based on the older age of patients, high-risk cytogenetics, and higher number of prior treatments. Allogeneic hematopoietic cell transplant (HCT) is the only potentially curative therapy available. Eight hundred and two adults with sAML and prior MDS/MPN who received a first HCT between 2000 and 2016 were included in the European Society for Blood and Marrow Transplant (EBMT) Acute Leukemia Working Party (ALWP) study. Median age of the cohort was 59.6 years (range, 18.6-78.6 years). Myeloablative conditioning (MAC) was given to 40% of patients, and 60% received reduced-intensity conditioning (RIC). Overall, the 2-year cumulative incidence of relapse (RI) was 37%, leukemia-free survival (LFS) was 40%, overall survival (OS) was 46%, nonrelapse mortality (NRM) was 23%, and chronic graft-versus-host disease (cGVHD) was 39%. In univariate analysis, a statistical difference between conditioning regimens 6 months after HCT in favor of the MAC group was noted with regard to RI (hazard ratio [HR], 1.47; P = .03), LFS (HR, 1.43; P = .01), and OS (HR, 1.55; P < .05). There was no difference in the cumulative incidence of NRM (HR, 1.38; P = .15). This effect was similarly seen in multivariate analysis (MVA): cumulative incidence of relapse (HR, 1.79; P < .05), LFS (HR, 1.43; P = .02), and OS (HR, 1.53; P = .005) with no difference in NRM (HR, 1; P = .98). This EBMT ALWP analysis suggests that long-term survival can be achieved in patients with sAML with antecedent MDS/MPN and that MAC is a suitable conditioning regimen in patients with sAML.

Abstract: The only curative treatment in patients with intermediate or high-risk myelodysplastic syndrome (MDS) is allogeneic hematopoietic stem cell transplantation (HSCT), which usually results in a long-term, disease-free survival rate of between 30% and 50%, depending on the disease risk and the type of donor. In patients without an HLA-matched sibling donor, a family haploidentical donor is an alternative option. The present study reports the European Group for Blood and Marrow Transplantation activity for haploidentical transplantation in MDS patients. A total of 228 patients transplanted from a mismatched HLA-related donor between 2007 and 2014 were studied. The median age at transplant was 56 years. Eighty-four (37%) patients had MDS transformed into acute myeloid leukemia at the time of transplant. Ex vivo T-cell depletion was used in 34 patients. One hundred ninety-four patients received a T-cell replete transplant and 102 patients received posttransplant cyclophosphamide (PT-CY) as graft-versus-host disease (GVHD) prophylaxis. The cumulative incidences of acute and chronic GVHD in PT-CY vs other patients were 25% vs 37% and 37% vs 24%, respectively. The cumulative incidence of nonrelapse mortality was 55% in patients who did not receive PT-CY (no PT-CY) and 41% in patients who did receive PT-CY. Three-year overall survival was 28% in no PT-CY patients and 38% in PT-CY patients. In multivariable analysis, the main risk factors were the intensity of the conditioning regimen and the use of PT-CY. In conclusion, the outcomes of MDS patients who received an haploidentical transplant are close to the results other transplantations from HLA-mismatched donors with approximately one-third of patients alive and free of disease 3 years after transplant, and the use of PT-CY may improve their outcomes.

Dietary restriction can extend the life of various model organisms. Here, Xie et al. show that intermittent periods of fasting achieved through every-other-day feeding protect mice against neoplastic disease but do not broadly delay organismal aging in animals.

Abstract Background Primary refractory acute myeloid leukemia (PRF-AML) is associated with a dismal prognosis. Allogeneic stem cell transplantation (HSCT) in active disease is an alternative therapeutic strategy. The increased availability of unrelated donors together with the significant reduction in transplant-related mortality in recent years have opened the possibility for transplantation to a larger number of patients with PRF-AML. Moreover, transplant from unrelated donors may be associated with stronger graft-mediated anti-leukemic effect in comparison to transplantations from HLA-matched sibling donor, which may be of importance in the setting of PRF-AML. Methods The current study aimed to address the issue of HSCT for PRF-AML and to compare the outcomes of HSCT from matched sibling donors (n = 660) versus unrelated donors (n = 381), for patients with PRF-AML between 2000 and 2013. The Kaplan-Meier estimator, the cumulative incidence function, and Cox proportional hazards regression models were used where appropriate. Results HSCT provide patients with PRF-AML a 2-year leukemia-free survival and overall survival of about 25 and 30%, respectively. In multivariate analysis, two predictive factors, cytogenetics and time from diagnosis to transplant, were associated with lower leukemia-free survival, whereas Karnofsky performance status at transplant ≥90% was associated with better leukemia-free survival (LFS). Concerning relapse incidence, cytogenetics and time from diagnosis to transplant were associated with increased relapse. Reduced intensity conditioning regimen was the only factor associated with lower non-relapse mortality. Conclusions HSCT was able to rescue about one quarter of the patients with PRF-AML. The donor type did not have any impact on PRF patients’ outcomes. In contrast, time to transplant was a major prognostic factor for LFS. For patients with PRF-AML who do not have a matched sibling donor, HSCT from an unrelated donor is a suitable option, and therefore, initiation of an early search for allocating a suitable donor is indicated.

Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics.

The aim of this study was to develop and validate a clinical and transplant-specific prognostic score using data from a large cohort of patients with myelodysplastic syndromes reported to the European Society for Blood and Marrow Transplantation registry. A Cox model was fitted to detect clinical and transplant-related variables prognostic of outcome. Then, cross-validation was performed to evaluate the validity and consistency of the model. Seven independent risk factors for survival were identified: age ≥50 years, matched unrelated donor, Karnofsky Performance Status 1%, and platelet count ≤50 × 109/L prior to transplantation. Incorporating these factors into a four-level risk score yielded hazard ratios for death, with low-risk (score of 0-1) as reference, of 2.02 (95% CI: 1.41-2.90) for the intermediate-risk group (score of 2-3), 3.49 (95% CI: 2.45-4.97) for the high-risk group (score of 4-5), and 5.90 (95% CI: 4.01-8.67) for the very high-risk group (score of >5). The score was predictive of survival, relapse-free survival, relapse, and non-relapse mortality (P

Primary refractory or relapsed acute myeloid leukemia is associated with a dismal prognosis. Allogeneic stem cell transplantation is the only therapeutic option that offers prolonged survival and cure in this setting. In the absence of a matched sibling donor, transplantation from unrelated 10/10 HLA allele-matched or 9/10 HLA allele-mismatched donors and haploidentical donors are potential alternatives. The current study aimed to compare the outcomes of acute myeloid leukemia patients with active disease who received allogeneic stem cell transplantation from a haploidentical donor with post-transplant cyclophosphamide (n=199) versus an unrelated 10/10-matched donor (n=1111) and versus an unrelated 9/10-mismatched donor (n=383) between 2007 and 2014 and who were reported to the European Society for Blood and Marrow Transplantation registry. Propensity score weighted analysis was conducted in order to control for disease risk imbalances between the groups. The leukemia-free survival rates at 2 years of recipients of grafts from a haploidentical donor, an unrelated 10/10-matched donor and an unrelated 9/10-mismatched donor were 22.8%, 28% and 22.2%, respectively (P=NS). In multivariate analysis, there were no significant differences in leukemia-free survival, overall survival, relapse incidence, non-relapse mortality, or graft-versus-host-disease-free relapse-free survival between the three groups. Two predictive factors were associated with a higher relapse incidence: transplantation during first or second relapse compared to primary refractory acute myeloid leukemia and poor cytogenetics. Allogeneic stem cell transplantation may rescue about 25% of acute myeloid leukemia patients with active disease. Importantly, the outcomes of transplants from haploidentical donors were comparable to those from 10/10-matched and 9/10-mismatched unrelated donors. Therefore, a haploidentical donor is a valid option for acute myeloid leukemia patients with active disease.

Abstract Background Myeloablative (MAC) and reduced-intensity conditioning (RIC) are established approaches for allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML). Most deaths after MAC occur within the first 2 years after SCT, while patients surviving leukemia-free for 2 years can expect a favorable long-term outcome. However, there is paucity of data on the long-term outcome (beyond 10 years) and the pattern of late events following RIC due to the relative recent introduction of this approach. Methods We analyzed long-term outcomes in a cohort of 1423 AML patients, age ≥50 years, after SCT from HLA-matched siblings, during the years 1997–2005, median follow-up 8.3 years (0.1–17). Results The 10-year leukemia-free survival (LFS) was 31 % (95CI, 27–35) and 32 % (28–35) after MAC and RIC, respectively (P = 0.57). The 10-year GVHD/ relapse-free survival (GRFS), a surrogate for quality of life was 22 % (18–25) and 21 % (18–24), respectively (P = 0.79). The 10-year non-relapse mortality (NRM) was higher and relapse rate was lower after MAC, throughout the early and late post-transplant course. The 10-year LFS among 584 patients surviving leukemia-free 2 years after SCT was 71 % (65–76) and 73 % (67–78) after MAC and RIC, respectively (P = 0.76). Advanced leukemia at SCT was the major predictor of LFS subsequent to the 2-year landmark. Relapse was the major cause of late death after both regimens; however, NRM and in particular chronic graft-versus-host disease and second cancers were more common causes of late death after MAC. Conclusions Long-term LFS and GRFS are similar after RIC and MAC. Most events after RIC or MAC occur within the first 2 years after SCT. Patients who are leukemia-free 2 years after SCT can expect similar good subsequent outcome after both approaches.

Abstract Background Allogeneic stem cell transplantation is the only curative option for patients with acute myeloid leukemia (AML) experiencing relapse. Either matched sibling donor (MSD) or unrelated donor (UD) is indicated. Methods We analyzed 1554 adults with AML transplanted from MSD (n = 961) or UD (n = 593, HLA-matched 10/10, n = 481; 9/10, n = 112). Compared to MSD, UD recipients were older (49 vs 52 years, p = 0.001), transplanted more recently (2009 vs 2006, p = 0.001), and with a longer interval to transplant (10 vs 9 months, p = 0.001). Conditioning regimen was more frequently myeloablative for patients transplanted with a MSD (61 vs 46 %, p = 0.001). Median follow-up was 28 (range 3–157) months. Results Cumulative incidence (CI) of neutrophil engraftment (p = 0.07), grades II–IV acute GVHD (p = 0.11), chronic GVHD (p = 0.9), and non-relapse mortality (NRM, p = 0.24) was not different according to the type of donor. At 2 years, CI of relapse (relapse incidence (RI)) was 57 vs 49 % (p = 0.001). Leukemia-free survival (LFS) at 2 years was 21 vs 26 % (p = 0.001), and overall survival (OS) was 26 vs 33 % (p = 0.004) for MSD vs UD, respectively. Chronic GVHD as time-dependent variable was associated with lower RI (HR 0.78, p = 0.05), higher NRM (HR 1.71, p = 0.001), and higher OS (HR 0.69, p = 0.001). According to HLA match, RI was 57 vs 50 vs 45 %, (p = 0.001) NRM was 23 vs 23 vs 29 % (p = 0.26), and LFS at 2 years was 21 vs 27 vs 25 % (p = 0.003) for MSD, 10/10, and 9/10 UD, respectively. In multivariate analysis adjusted for differences between the two groups, UD was associated with lower RI (HR 0.76, p = 0.001) and higher LFS (HR 0.83, p = 0.001) compared to MSD. Interval between diagnosis and transplant was the other factor associated with better outcomes (RI (HR 0.62, p

Abstract Background Data comparing fully matched and mismatched-unrelated-donor (M- and mM-URD) allogeneic hematopoietic stem cell transplant (allo-SCT) following reduced intensity conditioning regimens for acute myeloid leukemia are limited. Methods We retrospectively compared the outcome of 3398 patients above the age of 50 years who underwent 10/10 M-URD (n = 2567), 9/10 (n = 723), or 8/10 (n = 108) mM-URD allo-SCT for acute myeloid leukemia after reduced intensity conditioning regimen between 2000 and 2013. The Kaplan-Meier estimator, the cumulative incidence function, and Cox proportional hazards regression models were used where appropriate. Results HLA matching had no impact on engraftment (p = 0.31). In univariate analysis, in comparison to 10/10 M-URD, mM-URD was associated with higher incidence of grade II–IV acute graft-versus-host disease (GVHD) (p = 0.0002), similar rates of chronic GVHD (p = 0.138) but increased incidence of its extensive form (p = 0.047). Compared to 10/10 M-URD, patients transplanted in the first complete remission (CR1) with a 9 or an 8/10 mM-URD had decreased 2-year leukemia free (LFS) (p = 0.005) and overall survivals (OS) (56.7, 46.1, and 50.2 %, respectively, p = 0.005), while outcomes were comparable between all groups for patients transplanted beyond CR1. In multivariate analysis, 9/10 versus 10/10 URD was associated with higher non-relapse mortality (HR 1.34, p = 0.001), similar risk of relapse and chronic GVHD and inferior LFS (HR 1.25, p = 0.0001), and OS (HR 1.27, p = 0.0001). There was no difference in adjusted transplant outcomes between 9/10 and 8/10 mM-URD. Conclusions Reduced intensity conditioned allo-SCT with a 10/10 M-URD remains the preferable option for AML patients above the age of 50 years. The use of a 9/10 or an 8/10 mM-URD in patients not having a fully matched donor represents an alternative therapeutic option that should be compared to other alternative donor transplant strategies.

Familial myelodysplastic syndromes arise from haploinsufficiency of genes involved in hematopoiesis and are primarily associated with early-onset disease. Here we describe a familial syndrome in seven patients from four unrelated pedigrees presenting with myelodysplastic syndrome and loss of chromosome 7/7q. Their median age at diagnosis was 2.1 years (range, 1–42). All patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without an increase of blasts. Genomic studies identified constitutional mutations (p.H880Q, p.R986H, p.R986C and p.V1512M) in the SAMD9L gene on 7q21, with decreased allele frequency in hematopoiesis. The non-random loss of mutated SAMD9L alleles was attained via monosomy 7, deletion 7q, UPD7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21. Incomplete penetrance was noted in 30% (3/10) of mutation carriers. Long-term observation revealed divergent outcomes with either progression to leukemia and/or accumulation of driver mutations (n=2), persistent monosomy 7 (n=4), and transient monosomy 7 followed by spontaneous recovery with SAMD9L-wildtype UPD7q (n=2). Dysmorphic features or neurological symptoms were absent in our patients, pointing to the notion that myelodysplasia with monosomy 7 can be a sole manifestation of SAMD9L disease. Collectively, our results define a new subtype of familial myelodysplastic syndrome and provide an explanation for the phenomenon of transient monosomy 7. Registered at: www.clinicaltrials.gov; #NCT00047268.

No standard exists for the treatment of myelodysplastic syndrome relapsing after allogeneic stem cell transplantation. We performed a retrospective registry analysis of outcomes and risk factors in 698 patients, treated with different strategies. The median overall survival from relapse was 4.7 months (95% confidence interval: 4.1–5.3) and the 2-year survival rate was 17.7% (95% confidence interval: 14.8–21.2%). Shorter remission after transplantation (P

The effects of erythropoietin on osteoblasts and bone formation are controversial. Since patients with myelodysplastic syndromes often display excessively high erythropoietin levels, we aimed to analyze the effect of erythropoietin on osteoblast function in myelodysplastic syndromes and define the role of Wnt signaling in this process. Expression of osteoblast-specific genes and subsequent osteoblast mineralization was increased in mesenchymal stromal cells from healthy young donors by in vitro erythropoietin treatment. However, erythropoietin failed to increase osteoblast mineralization in old healthy donors and in patients with myelodysplasia, whereas the basal differentiation potential of the latter was already significantly reduced compared to that of age-matched controls (P

The universal modular chimeric antigen receptor (UniCAR) platform redirects CAR-T cells using a separated, soluble targeting module with a short half-life. This segregation allows precise controllability and flexibility. Herein we show that the UniCAR platform can be used to efficiently target solid cancers in vitro and in vivo using a pre-clinical prostate cancer model which overexpresses prostate stem cell antigen (PSCA). Short-term administration of the targeting module to tumor bearing immunocompromised mice engrafted with human UniCAR-T cells significantly delayed tumor growth and prolonged survival of recipient mice both in a low and high tumor burden model. In addition, we analyzed phenotypic and functional changes of cancer cells and UniCAR-T cells in association with the administration of the targeting module to reveal potential immunoevasive mechanisms. Most notably, UniCAR-T cell activation induced upregulation of immune-inhibitory molecules such as programmed death ligands. In conclusion, this work illustrates that the UniCAR platform mediates potent anti-tumor activity in a relevant in vitro and in vivo solid tumor model.

A significant proportion of hematopoietic stem cell transplants are performed with ABO-mismatched donors. The impact of ABO mismatch on outcome following transplantation remains controversial and there are no published data regarding the impact of ABO mismatch in acute myeloid leukemia patients receiving haploidentical transplants. Using the European Blood and Marrow Transplant Acute Leukemia Working Group registry we identified 837 patients who underwent haploidentical transplantation. Comparative analysis was performed between patients who received ABO-matched versus ABO-mismatched haploidentical transplants for common clinical outcome variables. Our cohort consisted of 522 ABO-matched patients and 315 ABO-mismatched patients including 150 with minor, 127 with major, and 38 with bi-directional ABO mismatching. There were no significant differences between ABO matched and mismatched patients in terms of baseline disease and clinical characteristics. Major ABO mismatching was associated with inferior day 100 engraftment rate whereas multivariate analysis showed that bi-directional mismatching was associated with increased risk of grade II–IV acute graft-versus-host disease [hazard ratio (HR) 2.387; 95% confidence interval (CI): 1.22–4.66; P=0.01). Non-relapse mortality, relapse incidence, leukemia-free survival, overall survival, and chronic graft-versus-host disease rates were comparable between ABO-matched and -mismatched patients. Focused analysis on stem cell source showed that patients with minor mismatching transplanted with bone marrow grafts experienced increased grade II–IV acute graft-versus-host disease rates (HR 2.03; 95% CI: 1.00–4.10; P=0.04). Patients with major ABO mismatching and bone marrow grafts had decreased survival (HR=1.82; CI 95%: 1.048 – 3.18; P=0.033). In conclusion, ABO incompatibility has a marginal but significant clinical effect in acute myeloid leukemia patients undergoing haploidentical transplantation.

Recent treatments of leukemias with chimeric antigen receptor (CAR) expressing T cells underline their impressive therapeutic potential. However, once adoptively transferred into patients, there is little scope left to shut them down after elimination of tumor cells or in case adverse side effects occur. This becomes of special relevance if they are directed against commonly expressed tumor associated antigens (TAAs) such as receptors of the ErbB family. To overcome this limitation, we recently established a modular CAR platform technology termed UniCAR. UniCARs are not directed against TAAs but instead against a unique peptide epitope on engineered recombinant targeting modules (TMs), which guide them to the target. In the absence of a TM UniCAR T cells are inactive. Thus an interruption of any UniCAR activity requires an elimination of unbound TM and the TM complexed with UniCAR T cells. Elimination of the latter one requires a disassembly of the UniCAR-TM complexes. Here, we describe a first nanobody (nb)-based TM directed against EGFR. The novel TM efficiently retargets UniCAR T cells to EGFR positive tumors and mediates highly efficient target-specific and target-dependent tumor cell lysis both in vitro and in vivo. After radiolabeling of the novel TM with 64Cu and 68Ga, we analyzed its biodistribution and clearance as well as the stability of the UniCAR-TM complexes. As expected unbound TM is rapidly eliminated while the elimination of the TM complexed with UniCAR T cells is delayed. Nonetheless, we show that UniCAR-TM complexes dissociate in vitro and in vivo in a concentration-dependent manner in line with the concept of a repeated stop and go retargeting of tumor cells via the UniCAR technology.

The pathogenesis and progression of many tumors, including hematologic malignancies is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits accelerated proliferation of tumor cells by providing membrane components but these may also alter physicochemical properties of lipid bilayers, which can impact signaling or even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of individual tumors to be explored as diagnostic markers. We have used the shotgun MS approach to identify lipid patterns in different types of acute myeloid leukemia (AML) patients that either show no karyotype change or belong to t(8;21) or inv16 types. Differences in lipidomes of t(8;21) and inv(16) patients, as compared to AML patients without karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid synthesis. Furthermore, between the t(8;21) and all other patients we observed significant changes in physicochemical membrane properties. These were related to a marked alteration in lipid saturation levels. The discovered differences in lipid profiles of various AML types improve our understanding of the pathobiochemical pathways involved and may serve in the development of diagnostic tools.

Background: Oesophageal varices and gastrointestinal bleeding are common complications of liver cirrhosis. More rarely, oesophageal varices occur in patients with non-cirrhotic portal hypertension that results from thromboses of portal or splanchnic veins. Case Report: We describe 2 young men who initially presented with varices as a result of portal vein thromboses. In the clinical follow-up, both were tested positive for a JAK2 mutation and consequently diagnosed with myeloproliferative neoplasms (MPNs). In an attempt to characterise the frequency of gastrointestinal complications in patients with JAK2-positive MPNs, we retrospectively analysed all known affected patients from our clinic for the diagnosis of portal vein thromboses and oesophageal varices. Strikingly, 48% of those who had received an oesophagogastroduodenoscopy had detectable oesophageal or gastric varices, and 82% of those suffered from portal or splanchnic vein thromboses. Conclusion: While the association between JAK2, myeloproliferative disease and thrombotic events is well established, patients with idiopathic oesophageal varices are not regularly tested for JAK2 mutations. However, the occurrence of oesophageal varices may be the first presenting symptom of a MPN with a JAK2 mutation, and affected patients may profit from a close haematological monitoring to assure the early detection of developing MPN.

The outcome of patients undergoing HLA-matched unrelated donor allogeneic hematopoietic cell transplantation following reduced-intensity conditioning or myeloablative regimens is reported to be equivalent; however, it is not known if the intensity of the conditioning impacts outcomes after mismatched unrelated donor transplantation for acute myeloid leukemia. Eight hundred and eighty three patients receiving reduced-intensity conditioning were compared with 1041 myeloablative conditioning regimen recipients in the setting of mismatched unrelated donor transplantation. The donor graft was HLA-matched at 9/10 in 872 (83.8%) and at 8/10 in 169 (16.2%) myeloablative conditioning recipients, while in the reduced-intensity conditioning cohort, 754 (85.4%) and 129 (14.6%) were matched at 9/10 and 8/10 loci, respectively. Myeloablative conditioning regimen recipients were younger, 70% being

Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrometry methods to analyze the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy, granulocyte-colony stimulating factor (G-CSF) mobilized stem cell donors were analyzed. In this Tandem Mass Tag (TMT) 10-plex labelling–based approach, 2070 proteins were identified with 171 proteins differentially abundant in one or both cellular compartments. This proof-of-principle-study demonstrates the potential of mass spectrometry to detect differentially expressed proteins in two compartment fractions of the entire proteome of leukemic stem cells, compared to their non-malignant counterparts. This may contribute to future immunotherapeutic target discoveries and individualized AML patient characterization.

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3′UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3′UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodys-plastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche.

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.

There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs). Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs) will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated) CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.

Population-specific matching probabilities (MP) are a key parameter to assess the benefits of unrelated stem cell donor registries and the need for further donor recruitment efforts. In this study, we describe a general framework for MP estimations of specific and mixed patient populations under consideration of international stem cell donor exchange. Calculations were based on population-specific 4-locus (HLA-A, -B, -C, -DRB1) high-resolution haplotype frequencies (HF) of up to 21 populations. In various scenarios, we calculated several quantities of high practical relevance, including the maximal MP that can be reached by recruiting a fixed number of donors, the corresponding optimal composition by population of new registrants, and the minimal number of donors who need to be recruited to reach a defined MP. Starting at current donor numbers, the largest MP increases due to n = 500,000 additional same-population donors were observed for patients from Bosnia-Herzegovina (+0.25), Greece (+0.21) and Romania (+0.20). Especially small MP increases occurred for European Americans (+0.004), Germans (+0.01) and Hispanic Americans (+0.01). Due to the large Chinese population, the optimal distribution of n = 5,000,000 new donors worldwide included 3.9 million Chinese donors. As a general result of our calculations, we observed a need for same-population donor recruitment in order to increase population-specific MP efficiently. This result was robust despite limitations of our input data, including the use of HF derived from relatively small samples ranging from n = 1028 (Bosnia-Herzegovina) to n = 33,083 (Turkey) individuals. National strategies that neglect domestic donor recruitment should therefore be critically re-assessed, especially if only few donors have been recruited so far.

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P

Simultaneous positron emission tomography (PET) and magnetic resonance imaging (MRI) is a new whole-body hybrid PET/MR imaging technique that combines metabolic and cross-sectional diagnostic imaging. Since the use of MRI in imaging of soft-tissue sarcoma is extremely beneficial, investigation of the combined PET/MRI is of great interest. In this paper, we present three cases and first data. Combined PET/MRI technique can support the process of clinical decision-making and give answers to some meaningful questions when treating patients with STS. Therefore, the combined modality of simultaneous PET/MRI offers new pieces to the puzzle of sarcoma treatment.

Regional HLA frequency differences are of potential relevance for the optimization of stem cell donor recruitment. We analyzed a very large sample (n = 123,749) of registered Polish stem cell donors. Donor figures by 1-digit postal code regions ranged from n = 5,243 (region 9) to n = 19,661 (region 8). Simulations based on region-specific haplotype frequencies showed that donor recruitment in regions 0, 2, 3 and 4 (mainly located in the south-eastern part of Poland) resulted in an above-average increase of matching probabilities for Polish patients. Regions 1, 7, 8, 9 (mainly located in the northern part of Poland) showed an opposite behavior. However, HLA frequency differences between regions were generally small. A strong indication for regionally focused donor recruitment efforts can, therefore, not be derived from our analyses. Results of haplotype frequency estimations showed sample size effects even for sizes between n≈5,000 and n≈20,000. This observation deserves further attention as most published haplotype frequency estimations are based on much smaller samples.

Background. Pleomorphic sarcoma is an aggressive soft tissue sarcoma. In patients with high-risk extremity sarcomas, the significant survival benefits conferred by an intense regimen of neoadjuvant chemoradiotherapy and surgery were reported. To our knowledge, this is the first report in the literature of the neoadjuvant use of trabectedin in a patient with high-grade pleomorphic sarcoma, ineligible for standard neoadjuvant combination therapy with an anthracycline-based regimen. Case Presentation. Here we present a 58-year-old White male with a large tumor in the left thigh, but with no signs of metastases. Owing to the history of severe heart attack, three cycles of neoadjuvant trabectedin were administrated to achieve surgically wide margins. After two cycles, an 18F-FDG-PET showed a large proportion of the central tumor area was without metabolic activity. According to RECIST and Choi criteria, the tumor was stable. After the third cycle of trabectedin, the patient underwent a complete resection, which revealed completely necrotic high-grade pleomorphic sarcoma (stage pT2b), with only a small vital area. Conclusion. The present paper on a promising treatment with neoadjuvant trabectedin of patients with high-grade pleomorphic sarcoma might suggest that such treatment approach may provide a greater chance of cure and survival of such patients.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.

BACKGROUND: Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC. METHODS: 47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha). RESULTS: EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01). CONCLUSIONS: EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.

Background According to WHO 2008 guidelines, the required percentage of cells manifesting dysplasia in the bone marrow to qualify as significant is 10% or over in one or more hematopoietic cell lineages, but this threshold is controversial. No ‘normal’ values have been established. Therefore, we investigated dyshematopoiesis in bone marrow aspirate squash preparations of 120 healthy bone marrow donors.Design and Methods Bone marrow squash slides of 120 healthy unrelated bone marrow donors were examined independently by 4 experienced morphologists. Samples were taken from the first aspiration during the harvest. Bone marrow preparation and assessment were performed according to WHO recommendations and ICSH guidelines.Results More than 10% dysmyelopoiesis could be detected in 46% of bone marrow aspirate squash preparations with 26% in 2 or more cell lineages and 7% in 3 cell lineages in healthy bone marrow donors. Donors under the age of 30 years exhibited more dysgranulopoietic changes and dysmegakaryopoietic changes (P

Background In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34+ cells and mesenchymal stromal cells with regard to different spatial localizations.Design and Methods Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay.Results In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner.Conclusions We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.