FLT3-directed UniCAR T-cell therapy of acute myeloid leukaemia.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape, which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform 'UniCAR' is currently under early clinical investigation. Recently, the first proof of concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off switch of UniCAR T cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T cells for the therapy of AML.

Intermediate-dose cytarabine plus mitoxantrone versus standard-dose cytarabine plus daunorubicin for acute myeloid leukemia in elderly patients.

Background: The combination of intermediate-dose cytarabine plus mitoxantrone (IMA) can induce high complete remission rates with acceptable toxicity in elderly patients with acute myeloid leukemia (AML). We present the final results of a randomized-controlled trial comparing IMA with the standard 7 + 3 induction regimen consisting of continuous infusion cytarabine plus daunorubicin (DA). Patients and methods: Patients with newly diagnosed AML >60 years were randomized to receive either intermediate-dose cytarabine (1000 mg/m2 twice daily on days 1, 3, 5, 7) plus mitoxantrone (10 mg/m2 days 1-3) (IMA) or standard induction therapy with cytarabine (100 mg/m2 continuously days 1-7) plus daunorubicin (45 mg/m2 days 3-5) (DA). Patients in complete remission after DA received intermediate-dose cytarabine plus amsacrine as consolidation treatment, whereas patients after IMA were consolidated with standard-dose cytarabine plus mitoxantrone. Results: Between February 2005 and October 2009, 485 patients were randomized; 241 for treatment arm DA and 244 for IMA; 76% of patients were >65 years. The complete response rate after DA was 39% [95% confidence interval (95% CI): 33-45] versus 55% (95% CI: 49-61) after IMA (odds ratio 1.89, P = 0.001). The 6-week early-death rate was 14% in both arms. Relapse-free survival curves were superimposable in the first year, but separated afterwards, resulting in 3-year relapse-free survival rates of 29% versus 14% in the DA versus IMA arms, respectively (P = 0.042). The median overall survival was 10 months in both arms (P = 0.513). Conclusion: The dose escalation of cytarabine in induction therapy lead to improved remission rates in the elderly AML patients. This did not translate into a survival advantage, most likely due to differences in consolidation treatment. Thus, effective consolidation strategies need to be further explored. In combination with an effective consolidation strategy, the use of intermediate-dose cytarabine in induction may improve curative treatment for elderly AML patients.

Allogeneic hematopoietic cell transplantation in intermediate risk acute myeloid leukemia negative for FLT3-ITD, NPM1- or biallelic CEBPA mutations.

BACKGROUND: The value of allogeneic hematopoietic cell transplantation (alloHCT) as postremission treatment is not well defined for patients with intermediate-risk acute myeloid leukemia (AML) without FLT3-ITD, biallelic CEBPA-, or NPM1 mutations (here referred to as NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML) in first complete remission (CR1). PATIENTS AND METHODS: We addressed this question using data from two prospective randomized controlled trials on intensive induction- and risk-stratified postremission therapy. The NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML subgroup comprised 497 patients, aged 18-60 years. RESULTS: In donor versus no-donor analyses, patients with a matched related donor had a longer relapse-free survival (HR 0.5; 95% CI 0.3-0.9, P = 0.02) and a trend toward better overall survival (HR 0.6, 95% CI 0.3-1.1, P = 0.08) compared with patients who received postremission chemotherapy. Notably, only 58% of patients in the donor group were transplanted in CR1. We therefore complemented the donor versus no-donor analysis with multivariable Cox regression analyses, where alloHCT was tested as a time-dependent covariate: overall survival (HR 0.58, 95% CI 0.37-0.9, P = 0.02) and relapse-free survival (HR 0.51, 95% CI 0.34-0.76; P = 0.001) for patients who received alloHCT compared with chemotherapy in CR1 were significantly longer. CONCLUSION: Outside clinical trials, alloHCT should be the preferred postremission treatment of patients with intermediate risk NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML in CR1. CINICALTRIALS.GOV IDENTIFIER: NCT00180115, NCT00180102.

T cell receptor repertoires after adoptive transfer of expanded allogeneic regulatory T cells.

Regulatory T cell (Treg ) therapy has been exploited in autoimmune disease, solid organ transplantation and in efforts to prevent or treat graft-versus-host disease (GVHD). However, our knowledge on the in-vivo persistence of transfused Treg is limited. Whether Treg transfusion leads to notable changes in the overall Treg repertoire or whether longevity of Treg in the periphery is restricted to certain clones is unknown. Here we use T cell receptor alpha chain sequencing (TCR-α-NGS) to monitor changes in the repertoire of Treg upon polyclonal expansion and after subsequent adoptive transfer. We applied TCR-α-NGS to samples from two patients with chronic GVHD who received comparable doses of stem cell donor derived expanded Treg . We found that in-vitro polyclonal expansion led to notable repertoire changes in vitro and that Treg cell therapy altered the peripheral Treg repertoire considerably towards that of the infused cell product, to different degrees, in each patient. Clonal changes in the peripheral blood were transient and correlated well with the clinical parameters. We suggest that T cell clonotype analyses using TCR sequencing should be considered as a means to monitor longevity and fate of adoptively transferred T cells.

[Myelodysplastic syndrome, acute leukemia and stem cell transplantation]. / Myelodysplastisches Syndrom, akute Leukämie und Stammzelltransplantation.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem cell disorders. They are characterized by inefficient hematopoiesis leading to peripheral cytopenia of one or more lineages and a variable risk of transformation into acute myeloid leukemia. They may either arise de novo as well as following exposition to environmental toxins, previous radiotherapy or chemotherapy or in the context of autoinflammatory diseases and related therapy. Characteristic cytogenetic abnormalities, along with the numbers of hematopoietic lineages affected and bone marrow blasts, enable an assessment of the risk of leukemic transformation. Acute leukemias are characterized by an accumulation of immature myeloid or lymphatic progenitor cells with limited differentiation capacity in the bone marrow. Proliferation of blast cells leads to suppression of normal hematopoiesis resulting in peripheral pancytopenia or leukocytosis associated with anemia and thrombocytopenia. Acute leukemias following MDS are defined as high-risk diseases. Intensive induction therapy followed by allogeneic stem cell transplantation is currently regarded as the only potentially curative treatment strategy. In this article the basic aspects of current diagnostics and treatment strategies for MDS and acute leukemia are outlined. Because of similarities with rheumatic inflammatory diseases, manifestations and treatment of graft versus host disease (GvHD) are also included.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αß sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting.

Feasibility of an exercise programme in elderly patients undergoing allogeneic stem cell transplantation - a pilot study.

It has been demonstrated that physical exercise benefits younger patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT). We designed a prospective pilot study investigating whether elderly patients (>60 years) would also be able to participate in such a programme. It consisted of physiotherapist-supervised alternating endurance and resistance workouts on 6 of 7 days a week. Sixteen consecutive patients undergoing allo-HSCT were enrolled into the study. The median age was 64.5 years. Twelve patients participated in the programme until the time of discharge (75%) from the transplant unit. Therefore, the predefined criteria regarding feasibility were met. The reason for drop out was transplantation associated mortality in all patients (n = 4). Adherence was very good with a median of 85% attended training sessions. No adverse events were recorded. The endurance capacity dropped by 7% and lower extremity strength improved by 2% over time. Quality of life decreased during the study period, with global health being significantly worse at the time of discharge. In conclusion, a combined and intensified strength and endurance exercise programme is feasible and safe in a population of elderly patients undergoing allo-HSCT. Further research should focus on exploring effect sizes of such an intervention by conducting randomised controlled trials.

Mito-FLAG with Ara-C as bolus versus continuous infusion in recurrent or refractory AML--long-term results of a prospective randomized intergroup study of the East German Study Group Hematology/Oncology (OSHO) and the Study Alliance Leukemia (SAL).

BACKGROUND: For patients with primary refractory or relapsed acute myeloid leukemia (AML), no treatment of choice has until now been defined to date. Cytarabine (Ara-C) is a key drug in the treatment of AML patients, there is still uncertainly regarding its optimal dose and infusion schedule. The aim of this study is to examine the impact of the Ara-C infusion schedule used as part of an intensive salvage regimen, in patients with relapsed or refractory AML. PATIENTS AND METHODS: A total of 252 adult patients (median age 59 years) with relapsed or refractory AML were randomly allocated to receive either Mito-FLAG with Ara-C as bolus (B) (1000 mg/m(2) over 1 h, every 12 h, days 1-5), or continuous infusion (CI) (150 mg/m(2) over 24 h, days 1-5) in combination with mitoxantrone, fludarabine, and granulocyte colony-stimulating factor (G-CSF). Autologous or allogeneic hematopoietic stem-cell transplantation was offered as consolidation therapy. Primary end point was the rate of complete remissions (CRs) after the first cycle of Mito-FLAG. RESULTS: The CR rates after Mito-FLAG (B) and Mito-FLAG (CI) were 54% and 43%, respectively (P = 0.1). There was no statistical difference between rates of grade 3/4 neutropenia, thrombocytopenia, mucositis, renal, and liver toxicity. More infections occurred, however, after Mito-FLAG (B) compared with Mito-FLAG (CI) (80% versus 69%, P = 0.01). The early death rate by day 42 was 13% in both arms. Median disease-free survival was comparable in the two arms (7.8 versus 7.1 months, P = 0.53) as was overall survival (7.1 versus 6.6 months, P = 0.53). CONCLUSION: A 5-day course of Ara-C 2 × 1000 mg/m(2) administered as bolus versus Ara-C 150 mg/m(2) administered by CI (in combination with mitoxantrone, fludarabine, and G-CSF), resulted in a nonsignificant trend in response rates in favor of Mito-FLAG (B) at the selected dose levels, but no differences in the survival outcome in relapsed or refractory AML. CLINICAL TRIAL NUMBER: LN_NN_2004_39/EudraCT number 2014-000083-18.

Methodological and clinical aspects of alloimmunization after granulocyte transfusion in patients undergoing allogeneic stem cell transplantation.

In allogeneic hematopoietic stem cell transplantation (HSCT), granulocyte transfusions (GT) may be required in immunocompromised, neutropenic patients. In this context, alloimmunization against alloantigens may occur and affect HSCT outcome. Anti-human leukocyte antigen (HLA) and -MHC class I chain related antigens A (MICA) antibody response after the administration of GT in 29 patients undergoing allogeneic HSCT (n = 27) encompassing 109 sera was investigated by multianalyte microbead assay before and up to 6 month after HSCT. Anti-HLA class I and II antibodies emerged de novo in 11 (38%) and 4 (14%) patients, respectively. Similarly, preformed antibodies were observed in four cases (14%) for anti-HLA class I and also four patients for anti-HLA class II antibodies. Anti-MICA antibodies were observed in eight granulocyte recipients of which three patients developed anti-MICA antibodies after GT, whereas preformed antibodies were seen in five patients. The conversion to positivity for any of the investigated antibodies did not significantly affect overall survival or the incidence of GVHD. GT-associated alloantibody conversion observed did not significantly correlate with outcome. Thus, surveillance of anti-HLA antibodies in the course of GT in the context of HSCT may not be required routinely. The role of MICA antibodies in HSCT and GT, however, requires further study.

Geriatric assessment and the variance of treatment recommendations in geriatric patients with gastrointestinal cancer-a study in AIO oncologists.

BACKGROUND: Geriatric assessment (GA) is recommended to detect vulnerabilities for elderly cancer patients. To assess whether results of GA actually influence the treatment recommendations, we conducted a case vignette-based study in medical oncologists. MATERIALS AND METHODS: Seventy oncologists gave their medical treatment recommendations for a maximum of 4 out of 10 gastrointestinal cancer patients in three steps: (i) based on tumor findings alone to simulate the guideline recommendation for a '50-year-old standard patient without comorbidities'; (ii) for the same situation in elderly patients (median age 77.5 years) according to the comorbidities, laboratory values and a short video simulating the clinical consultation; and (iii) after the results of a full GA including interpretation aid [Barthel Index, Cumulative Illness Rating Scale (CIRS), Geriatric 8 (G8), Geriatric Depression Scale (GDS), Mini Mental Status Examination (MMSE), Mini-Nutritional Assessment (MNA), Timed Get Up and Go (TGUG), European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-30 (EORTC QLQ-C30), stair climb test]. RESULTS: Data on 164 treatment recommendations were analyzed. The recommendations had a significantly higher variance for elderly patients than for 'standard' patients (944 versus 602, P < 0.0001) indicating a lower agreement between oncologists. Knowledge on GA had marginal influence on the treatment recommendation or its variance (944 versus 940, P = 0.92). There was no statistically significant influence of the working place or the years of experience in oncology on the variance of recommendations. The geriatric tools were rated approximately two times higher as being 'meaningful' (53%) and 'useful for the presented cases' (49%) than they were 'used in clinical practice' (19%). The most commonly used geriatric tool in patient care was the MNA (30%). CONCLUSIONS: The higher variance of treatment recommendations indicates that it is less likely for elderly patients to get the optimal recommendation. Although the proposed therapeutic regimen varied higher in elderly patients and the oncologists rated the GA results as 'useful', the GA results did not influence the individual recommendations or its variance. Continuing education on GA and research on implementation into clinical practice are needed.

IDO1 and IDO2 gene expression analysis by quantitative polymerase chain reaction.

Immunomodulatory properties of IDO1 relate to tryptophan catabolism. The degradation of tryptophan by IDO1 leads to suppression of T cell responses. Recently, another enzyme with IDO-like activity, indoleamine 2,3-dioxygenase-like-protein 1 (INDOL1, IDO2), has been described in both mice and humans. In order to study the gene expression of IDO1 and IDO2, we have developed a quantitative PCR (qPCR) assay. In an exploratory application to the study of the differential expression of IDO1 and IDO2 by professional antigen-presenting cells and MSCs (mesenchymal stromal cells) under the influence of interferon-γ (IFN-γ) and T-lymphocyte conditioned media (TCM), substantial differences were observed. IDO expression measured by qPCR was valid and reliable in the cell types investigated. Further studies are needed to delineate factors driving IDO expression in MSCs.

Trends in allogeneic haematopoietic cell transplantation for myelofibrosis in Europe between 1995 and 2018: a CMWP of EBMT retrospective analysis.

We performed a retrospective assessment of patient- and transplant-specific characteristics and outcomes for 4142 patients undergoing allogeneic haematopoietic cell transplant for myelofibrosis between 1995 and 2018 across 278 centres. Activity increased steadily across the four analysed eras (<2006, 2006-2010, 2011-2014 and 2015-2018). Median recipient age increased over time between the earliest and most recent cohort (49.4 years (range, 20.1-68) versus 59.3 years (range, 18.1-78.1). Increasing number of patients with a Karnofsky performance status <90 underwent transplant over time. Increased utilisation of matched unrelated donors was apparent (<2006, 22.5% versus 2015-18, 45.2%; p < 0.001). Decreased use of myeloablative conditioning, increased use of busulphan-based platforms and anti-thymocyte globulin was evident. Of note, rates of acute (a)GVHD grade II-IV by day +100 decreased over time (p = 0.027) as did rates of chronic (c) GVHD, predominantly extensive cGVHD (<2006, 36% (31-41%) versus 2015-18, 23% (21-25%); p = 0.001). Overall, significant factors associated with worse overall survival and non-relapse mortality (NRM) remained older age, use of donors other than matched sibling, recipient CMV seropositivity and a lower Karnofsky performance status (<90). Multivariable analysis demonstrated improvements in overall survival and reductions in relapse risk over time with stable NRM rates despite increasing numbers of older, less fit patients and use of unrelated donors.

Solid organ transplantation after allogeneic hematopoietic stem cell transplantation: a retrospective, multicenter study of the EBMT.

To analyze the outcome of solid organ transplantation (SOT) in patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT), a questionnaire survey was carried out within 107 European Group of Blood and Marrow Transplantation centers. This study covered HSCT between 1984 and 2007 in Europe. Forty-five SOT in 40 patients were reported. Fifteen liver, 15 renal, 13 lung, 1 heart and 1 skin transplantations were performed in 28 centers. Overall survival (OS) of patients after SOT was 78% at 5 years (95% confidence interval [CI], 64% to 92%). OS at 5 years was 100% for renal, 71% (95% CI, 46% to 96%) for liver and 63% (95% CI, 23% to 100%) for lung transplant recipients. The 2-year-incidence of SOT failure was 20% (95% CI, 4% to 36%) in patients with graft-versus-host disease (GvHD) and 7% (95% CI, 0% to 21%) in patients without GvHD before SOT. The relapse incidence for underlying malignant diseases was 4% at 5 years (95% CI, 0% to 12%). In summary, this study shows that selected patients receiving SOT after HSCT have a remarkably good overall and organ survival. These data indicate that SOT should be considered in selected patients with single organ failure after HSCT.

[Bone tissue engineering in clinical application : assessment of the current situation]. / Knochen-Tissue-Engineering in der klinischen Anwendung : Eine Standortbestimmung.

Treatment of severe bone defects remains a challenge in orthopaedic surgery and traumatology. Surgical techniques should provide primary stability to reach osseous integration and secondary remodeling of bone grafts and substitute materials. None of the currently available substitute materials provides osteoconduction and osteogenesis comparable to those of human allografts and autografts. To enhance osteoinductive and osteogenetic properties of these implants mesenchymal stem cells are used successfully in bone tissue engineering approaches. The aim of this report is to summarize the currently available data on bone tissue engineering and preliminary experience with a tissue engineered graft in acetabular revision surgery after loosening of a hip replacement.

Net reclassification improvement with serial biomarkers and bed-sided spirometry to early predict the need of organ support during the early post-transplantation in-hospital stay in allogeneic HCT recipients.

To predict the need of intensive care unit admission with organ support during the transplantation hospital stay in 101 consecutives allogeneic hematopoietic cell transplantation (allo-HCT) recipients the added predictive utility of three times per week Copeptin, MR-proADM, MR-proANP, NT-proBNP, IL-6, Procalcitonin, D-dimer and three times per week bed-sided pulmonary function test was determined in comparison with an index model. The index model was calculated by multivariate regression analysis out of the patients' routine laboratory parameters. To calculate the added predictive utility of the investigated markers the Δ-AUC and the continuous net reclassification improvement (cNRI + 2 to - 2), splitted for events and non-events were calculated for each marker in comparison with the index model. According to the Δ-AUC, none of the parameters improved risk prediction. In contrast, the cNRI was significantly improved for events and non-events by Copeptin (event 0.75, p value 0.0013; non-event 0.4, p value 0.000079) and for events by NT-proBNP (0.6, p value 0.018). D-dimer and PCT significantly predicted the non-event. Of the spirometry parameters, the FEF50% improved prediction of event and non-event according to the cNRI model. Our data support the additional serial analysis of Copeptin and NT-proBNP in allo-HCT recipients during the transplantation hospital stay.

Targeting mycophenolate mofetil for graft-versus-host disease prophylaxis after allogeneic blood stem cell transplantation.

As low trough levels of mycophenolic acid (MPA) have been measured in recipients of allo-SCTs, we performed a pilot study targeting mycophenolate mofetil (MMF) doses according to the MPA area under the concentration (AUC) levels. Twenty-nine patients were transplanted from matched sibling (n=7) and unrelated donors (n=22). Tacrolimus was given orally from day -1 to achieve trough blood levels of 5-10 ng/ml. MMF was started on day 0 at 1500 mg intravenously b.i.d. AUC measurements of MPA by HPLC were scheduled on days 3, 7 and 11 after transplantation. The MMF dose was modified to achieve an MPA AUC of 35-60 microg/ml/h. With the respective adjustments 66 and 75% surpassed the lower AUC target on days 7 and 11, respectively. The cumulative incidence of grade III-IV acute GVHD was 28% (8/29). Eight out of 24 evaluable patients (33%) suffer from limited (n=3) or extensive (n=5) chronic GVHD. Overall, the results of this study suggest that targeting of MPA exposure is feasible early after transplantation. A simplified MMF targeting strategy based on MPA C(max) or C(2h) levels seems to be warranted in future trials involving more patients at later time points in the outpatient setting.

Bortezomib significantly impairs the immunostimulatory capacity of human myeloid blood dendritic cells.

Bortezomib is a potent drug for the treatment of multiple myeloma. Its anti-tumor activity is mediated by proteasome inhibition leading to decreased cell proliferation and induction of apoptosis. However, an unimpaired proteasomal function plays a crucial role for the induction of anti-tumor immunity by dendritic cells (DCs), which are currently used for therapeutic vaccination against various tumors including myeloma. In the present study, we investigated the impact of bortezomib on the immunostimulatory capacity of 6-sulfo LacNAc (slan) DCs, which represent a major subset of human blood DCs. We demonstrated that this proteasome inhibitor efficiently impairs the spontaneous in vitro maturation of slanDCs and the release of tumor necrosis factor (TNF)-alpha as well as interleukin (IL)-12 upon lipopolysaccharide (LPS) stimulation. Functional data revealed that bortezomib profoundly inhibits slanDC-induced proliferation and differentiation of CD4(+) T cells. In addition, the capacity of slanDCs to promote interferon-gamma secretion and tumor-directed cytotoxicity of natural killer (NK) cells is markedly impaired by bortezomib. These results provide evidence that bortezomib significantly reduces the ability of native human blood DCs to regulate innate and adaptive anti-tumor immunity and may have implications for the design of therapeutic strategies combining DC vaccination and bortezomib treatment.

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants.

Monitoring of minimal residual disease (MRD) has become an important clinical aspect for early relapse detection during follow-up care after cancer treatment. Still, the sensitive detection of single base pair point mutations via Next-Generation Sequencing (NGS) is hampered mainly due to high substitution error rates. We evaluated the use of NGS for the detection of low-level variants on an Ion Torrent PGM system. As a model case we used the c.1849G > T (p.Val617Phe) mutation of the JAK2-gene. Several reaction parameters (e.g. choice of DNA-polymerase) were evaluated and a comprehensive analysis of substitution errors was performed. Using optimized conditions, we reliably detected JAK2 c.1849G > T VAFs in the range of 0.01-0.0015% which, in combination with results obtained from clinical data, validated the feasibility of NGS-based MRD detection. Particularly, PCR-induced transitions (mainly G > A and C > T) were the major source of error, which could be significantly reduced by the application of proofreading enzymes. The integration of NGS results for several common point mutations in various oncogenes (i.e. IDH1 and 2, c-KIT, DNMT3A, NRAS, KRAS, BRAF) revealed that the prevalent transition vs. transversion bias (3.57:1) has an impact on site-specific detection limits of low-level mutations. These results may help to select suitable markers for MRD detection and to identify individual cut-offs for detection and quantification.

Inhibition of platelet-derived growth factor receptorbeta by imatinib mesylate suppresses proliferation and alters differentiation of human mesenchymal stem cells in vitro.

OBJECTIVES: Recent data show that Imatinib mesylate (IM) also affects haematopoietic stem cells (HSC), T lymphocytes and dendritic cells that do not harbour constitutively active tyrosine kinases. MATERIALS AND METHODS: We evaluated possible effects of IM on human bone marrow-derived mesenchymal stem cells (MSC) in vitro. RESULTS: Screening the activity of 42 receptor tyrosine kinases revealed an exclusive inhibition of platelet-derived growth factor receptorbeta (PDGFRbeta). Analysis of downstream targets of PDGFRbeta demonstrated IM-mediated reduction of Akt and Erk1/2 phosphorylation. Culture of MSC with IM led to the reversible development of perinuclear multi-vesicular bodies. The proliferation and clonogenicity of MSC were significantly reduced compared to control cultures. IM favoured adipogenic differentiation of MSC whereas osteogenesis was suppressed. The functional deficits described led to a 50% reduction in the support of clonogenic haematopoietic stem cells, cultured for 1 month on a monolayer of MSC with IM. CONCLUSION: In summary, inhibition of PDGFRbeta and downstream Akt and Erk signalling by IM has a significant impact on proliferation and differentiation of human MSC in vitro.