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Abstract Background Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL). Methods Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 106 and 50 × 106 CARTs/m2. Leukapheresis, manufacturing and administration of CARTs were performed in-house. Results For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response. Conclusion In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www.clinicaltrials.gov as NCT03676504.

We retrospectively studied 97 acute myeloid leukemia patients with trisomy 19 (median age at diagnosis 57 years; range, 17- 83 years) treated between 2001 and 2019 within two multicenter study groups. Trisomy 19 occurred alone in ten (10.5%) patients, with additional abnormalities being present in non-complex karyotypes in eight (8%) patients and in complex karyotypes in 79 (82%) patients. Altogether, karyotypes characterized by trisomies only were present in 27 (28%) patients. Data on response and outcome of intensively treated patients were available for 92 cases. The median follow-up was 6.4 years (95% confidence interval [95% CI]: 2.9-9.0 years). The complete remission (CR) rate after induction therapy was 52% (48 patients); the early death rate was 10% (n=9). Notably, patients with trisomy 19 as the sole abnormality had a CR rate of 89%. Allogeneic hematopoietic stem cell transplantation (allo-HCT) was performed in 34 (35%) patients (CR, n=19; active disease, n=15). Five-year relapse-free and overall survival rates were 26% (95% CI: 16-43%) and 20% (95% CI: 13-31%), respectively. Overall survival rates were significantly higher in patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time-dependent covariable on overall survival revealed that trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only was a favorable factor (hazard ratio [HR]=0.47; P=0.021); higher age at diagnosis had an adverse impact (10 years difference; HR=1.29; P=0.002), whereas allo-HCT did not have a beneficial impact (odds ratio=1.45; P=0.21). In our cohort, patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only had a high CR rate and better clinical outcome.

We retrospectively studied 125 patients with acute myeloid leukemia and trisomy 4 (median age at diagnosis, 58 years; range, 16-77 years) treated between 2000 and 2019 within a multicenter study. Trisomy 4 was the sole abnormality in 28 (22%) patients and additional abnormalities were present in 97 (78%) patients. Twenty-two (22%) and 15 (15%) of 101 tested patients harbored NPM1 and FLT3-ITD mutations. Two (3%) of 72 tested patients had double CEBPA mutations. Data on response to intensive anthracycline-based induction therapy were available for 119 patients. Complete remission was achieved in 67% (n=80) and the early death rate was 5% (n=6). Notably, patients with trisomy 4 as sole abnormality had a complete remission rate of 89%. Allogeneic hematopoietic cell transplantation was performed in 40 (34%) patients, of whom 19 were transplanted in first complete remission. The median follow-up of the intensively treated cohort was 5.76 years (95% confidence interval [95% CI]: 2.99-7.61 years). The 5-year overall survival and relapse-free survival rates were 30% (95% CI: 22-41%) and 27% (95% CI: 18-41%), respectively. An Andersen-Gill regression model on overall survival revealed that favorable-risk according to the European LeukemiaNet classification (hazard ratio [HR]=0.34; P=0.006) and trisomy 4 as sole abnormality (HR=0.41; P=0.01) were favorable factors, whereas age with a difference of 10 years (HR=1.15; P=0.11), female gender (HR=0.74; P=0.20) and allogeneic hematopoietic cell transplantation (HR=0.64; P=0.14) did not have an significant impact. In our cohort, patients with trisomy 4 as their sole abnormality had a high complete remission rate and favorable clinical outcome. Allogeneic hematopoietic cell transplantation did not seem to improve overall survival.

Julia Franzen et al. investigate if changes in DNA methylation at specific genetic loci during cell culture expansion are due to a specific mechanism or gradual deregulation of an epigenetic state. Their results suggest that changes in CpG methylation are due to indirect epigenetic drift, rather than a consequence of targeting by DNA methyltransferases.

Acute lymphoblastic leukemia (ALL) can relapse in the extramedullary compartment, with or without medullary involvement. Response to treatment may be individual. We evaluated response to inotuzumab ozogamicin in 31 patients with relapsed/refractory B-ALL with extramedullary disease. Median age was 31 years (range, 19-81). All patients were heavily pretreated, including allogeneic hematopoietic stem cell transplantation (HSCT; n=18). Overall response rate after two cycles of inotuzumab ozogamicin was 84% (complete remission, 55%; partial remission, 29%; resistant disease, 13%; early death, 3%). The median follow-up was 29 months and median overall survival was 12.8 months. One-year and 2-year overall survival rates were 53% (95% CI: 37-76%) and 18% (95% CI: 8-43%), respectively. Age had no impact on overall survival when assessed as a continuous variable or dichotomized at 60 years. Twelve patients proceeded to allogeneic HSCT (complete remission, n=6; partial remission, n=3; resistant disease, n=3). Prior to allogeneic HSCT, eight patients received two or fewer cycles and four patients received three or four cycles of inotuzumab ozogamicin. Sinusoidal obstruction syndrome was reported in three patients, including one after transplantation. Allogeneic HSCT, evaluated as a time-dependent variable, had no impact on overall survival. Inotuzumab ozogamicin seems to be effective as a debulking strategy in relapsed/refractory ALL with extramedullary disease. However, inotuzumab ozogamicin followed by allogeneic HSCT seems not to be effective in maintaining long-term disease control.

There is a high need for novel treatment options in relapsed and refractory diffuse large B-cell lymphoma. Single agent mammalian target of rapamycin (mTOR) inhibitor treatment has shown promising efficacy in this entity. Here, we report on the results of the mTOR-inhibitor temsirolimus combined to standard rituximab-DHAP salvage regimen in a prospective, multicenter, phase II, open-label study. The STORM regimen consisted of rituximab 375 mg/m2 (day 2) and DHAP (dexamethasone 40 mg day 3-6, cisplatinum 100 mg/m2 day 3, cytarabine 2 × 2 g/m2 day 4) with temsirolimus added on day 1 and 8 of a 21-day cycle, with 2 to 4 cycles planned. In part I, dose levels of 25, 50, 75, and 100 mg for temsirolimus were predefined. Based on the observed toxicity profile, a temsirolimus dose of 25 mg was defined as recommended dose for the part II extension cohort of the trial. The intention-to-treat cohort comprised 53 patients. Median age was 63 years and median number of prior regimen was 1. All but 1 patient had prior rituximab exposure. Temsirolimus dose was 50 mg on day 1 and 8 in 6 patients from the part I of the trial and 25 mg in the remaining 47 patients. In general, treatment was well tolerated with leucopenia and thrombocytopenia as most frequent severe adverse events. The overall response rate after the last cycle of temsirolimus R-DHAP was 66% with 24% complete responses. The ability to mobilize stem cells was not impaired by the treatment regimen. Twenty-eight patients received consolidation treatment with high-dose therapy (HDT) and stem cell transplantation. Median duration of response was not reached. The total 2-year progression-free survival (PFS) and overall survival (OS) were 53% and 59%. Patients who were consolidated with HDT achieved a 2-year PFS and a 2-year OS of 77.8% and 82.1%, respectively. We conclude that temsirolimus can be safely added to rituximab and DHAP with promising activity.

The aim of this study was to evaluate the prognostic impact of FLT3-ITD in core-binding factor acute myeloid leukemia (CBFAML) in an international, multicenter survey of 97 patients of whom 52% had t(8;21)(q22;q22) and 48% had inv(16)(p13q22)/t(16;16)(p13;q22). The median age of the patients was 53 years (range, 19-81). Complete remission after anthracycline-based induction (n=86) and non-intensive therapy (n=11) was achieved in 97% and 36% of the patients, respectively. The median follow-up was 4.43 years (95% confidence interval [95% CI]: 3.35-7.39 years). The median survival after intensive and non-intensive treatment was not reached and 0.96 years, respectively. Among intensively treated patients, inv(16) with trisomy 22 (n=11) was associated with a favorable 4-year relapse-free survival rate of 80% (95% CI: 59-100%) as compared to 38% (95% CI: 27-54%; P=0.02) in all other patients with CBFAML/ FLT3-ITD (n=75). Overall, 24 patients underwent allogeneic hematopoietic cell transplantation (HCT), 12 in first complete remission and 12 after relapse. Allogeneic HCT in first complete remission was not beneficial (P=0.60); however, allogeneic HCT seemed to improve median survival in relapsed patients compared to that of patients treated with chemotherapy (not reached vs. 0.6 years, respectively; P=0.002). Excluding patients with inv(16) with trisomy 22, our data indicate that compathe outcome of CBF-AML patients with FLT3-ITD may be inferior to that of patients without FLT3-ITD (based on previously published data), suggesting that prognostically CBF-AML patients with FLT3-ITD should not be classified as favorable-risk. FLT3-inhibitors may improve the outcome of these patients.

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.

Ageing causes an inability to replace damaged tissue. Here, the authors perform proteomics analyses of human haematopoietic stem cells and other cells in the bone marrow niche at different ages and show changes in central carbon metabolism, reduced bone marrow niche function, and enhanced myeloid differentiation.

A higher capacity to grow under hypoxic conditions can lead to a more aggressive behavior of tumor cells. Determining tumor activity under hypoxia may identify chronic lymphocytic leukemia (CLL) with aggressive clinical course and predict response to chemo-immunotherapy (CIT). A metabolic score was generated by determining pyruvate kinase and lactate dehydrogenase, key enzymes of glycolysis, ex vivo in primary CLL samples under normoxic and hypoxic conditions. This score was further correlated with clinical endpoints and response to CIT in 96 CLL patients. 45 patients were classified as metabolic high risk (HR), 51 as low risk (LR). Treatment-free survival (TFS) was significantly shorter in HR patients (median 394 vs 723 days, p = .021). 15 HR patients and 14 LR patients received CIT after sample acquisition. HR patients had a significantly shorter progression-free survival after treatment compared to LR patients (median 216 days vs not reached, p = .008). Multivariate analysis evaluating age, IGHV, TP53 deletion or mutation and 11q22–23 deletion besides the capacity of tumor cells to grow under severe hypoxic conditions identified the metabolic profile as the strongest independent risk factor for shorter TFS (hazard ratio 2.37, p = .011). The metabolic risk can provide prognostic and predictive information complementary to genetic biomarkers and identify patients who might benefit from alternative treatment approaches. Keywords: CLL, Metabolism, PK M2, LDH, High-risk

Graft-vs.-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation, significantly affects the post-transplant morbidity and mortality. Systemic steroids remain the gold standard for the initial management of GvHD. However, up to 60% of patients will not sufficiently respond to steroids. Extracorporeal photopheresis (ECP), a cell-based immunotherapy, has shown good clinical results in such steroid-refractory/resistant GvHD patients. Given its immunomodulatory, but not global immunosuppressive and steroid-sparing capacity, ECP constitutes an attractive option. In the case of GvHD, the balance of immune cells is destroyed: effector cells are not any longer efficiently controlled by regulatory cells. ECP therapy may restore this balance. However, the precise mechanism and the impact of ECP on anti-viral/anti-leukemic function remain unclear. In this study, 839 ECP treatments were performed on patients with acute GvHD (aGvHD) and chronic GvHD (cGvHD). A comprehensive analysis of effector and regulatory cells in patients under ECP therapy included multi-parametric flow cytometry and tetramer staining, LuminexTM-based cytokine, interferon-γ enzyme-linked immunospot, and chromium-51 release assays. Gene profiling of myeloid-derived suppressor cells (MDSCs) was performed by microarray analysis. Immunologically, modulations of effector and regulatory cells as well as proinflammatory cytokines were observed under ECP treatment: (1) GvHD-relevant cell subsets like CD62L+ NK cells and newly defined CD19hiCD20hi B cells were modulated, but (2) quantity and quality of anti-viral/anti-leukemic effector cells were preserved. (3) The development of MDSCs was promoted and switched from an inactivated subset (CD33−CD11b+) to an activated subset (CD33+CD11b+). (4) The frequency of Foxp3+CD4+ regulatory T cells (Tregs) and CD24+CD38hi regulatory B cells was considerably increased in aGvHD patients, and Foxp3+CD8+ Tregs in cGvHD patients. (5) Proinflammatory cytokines like IL-1β, IL-6, IL-8, and TNF-α were significantly reduced. In summary, ECP constitutes an effective immunomodulatory therapy for patients with steroid-refractory/resistant GvHD without impairment of anti-viral/leukemia effects.

Monoclonal antibodies against the cell surface antigen CD38, e.g., isatuximab, daratumumab, or Mor202, have entered the therapeutic armamentarium in multiple myeloma due to single agent overall response rates of 29 vs. 36 vs. 31%, effectivity in combination regimen, e.g., with lenalidomide or bortezomib plus dexamethasone, and tolerable side effects. Despite clinical use, many questions remain. In this manuscript, we address three of these: first, upfront CD38 target-expression in AL-amyloidosis, monoclonal gammopathy of unknown significance (MGUS), asymptomatic, symptomatic, and relapsed multiple myeloma. Second, relation of CD38-expression to survival, disease stages, molecular entities, and high-risk definitions. Third, alternative splicing or lack of CD38-expression as potential mechanisms of upfront resistance. We assessed CD138-purified plasma cell samples from 196 AL-amyloidosis, 62 MGUS, 259 asymptomatic, 764 symptomatic, and 90 relapsed myeloma patients, including longitudinal pairs of asymptomatic/symptomatic (n = 34) and symptomatic/relapsed myeloma (n = 57) regarding interphase fluorescence in situ hybridization (n = 1,380), CD38-expression by gene expression profiling (n = 1,371), RNA-sequencing (n = 593), and flow cytometry (n = 800). Samples of normal bone marrow plasma cells (n = 10), memory B-cells (n = 9), polyclonal plasmablastic cells (n = 9), and human myeloma cell lines (n = 54) were used as comparators. CD38 was expressed in all malignant plasma cell samples, but significantly lower compared to normal plasma cells with small but significant downregulation in longitudinal sample pairs. Higher CD38 expression was associated with the presence of t(4;14) and high-risk according to the UAMS70-gene score, lower expression was associated with del17p13 and hyperdiploidy in symptomatic myeloma as well as t(11;14) in asymptomatic myeloma. Higher CD38-expression was associated with slower progression to symptomatic and relapsed myeloma and better overall survival in the latter two entities. CD38 expression, t(4;14), del17p13, and gain of 1q21 are independently prognostic in multivariate analysis. By contrast, high CD38-expression is associated with adverse survival in AL-amyloidosis. Regarding mechanisms of upfront anti-CD38-treatment resistance, lack of CD38-expression and alternative splicing of receptor binding-sites could be excluded. Here, of the two protein coding CD38-transcripts CD38-001 (eight-exon, full length) and CD38-005 (truncated), CD38-001 conveyed >97% of reads spanning the respective CD38 splice junction.

IntroductionTherapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (TN) vs. effector (TE) T cells, TN cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the TN/TE ratio of CART cells.Materials and methodsCART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays.ResultsIL-7/IL-15 preferentially induced differentiation into TN, stem cell memory (TSCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (TEM), CD56+ and CD4+ T regulatory (TReg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CARTN cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CARTN cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CARTN cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CARTN cells in untreated CLL patients. Final TN/TE ratio stayed 2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CARTN expansion.ConclusionUntreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of TN among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the TN/TE ratio within the CART product.

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse.

Chemotherapy in light chain amyloidosis aims to normalize the involved free light chain in serum, which leads to an improvement, or at least stabilization of organ function in most responding patients. We performed a prospective single center phase 2 trial with 50 untreated patients not eligible for high-dose treatment. The treatment schedule comprised 6 cycles of oral lenalidomide, melphalan and dexamethasone every 4 weeks. After 6 months, complete remission was achieved in 9 patients (18%), very good partial remission in 16 (32%) and partial response in 9 (18%). Overall, organ response was observed in 24 patients (48%). Hematologic and cardiac toxicities were predominant adverse events. Mortality at 3 months was low at 4% (n=2) despite the inclusion of 36% of patients (n=18) with cardiac stage Mayo 3. After a median follow-up of 50 months, median overall and event-free survival were 67.5 months and 25.1 months, respectively. We conclude that the treatment of lenalidomide, melphalan and dexamethasone is very effective in achieving a hematologic remission, organ response and, consecutively, a long survival in transplant ineligible patients with light chain amyloidosis. However, as toxicity and tolerability are the major problems of a 3-drug regimen, a strict surveillance program is necessary and sufficient to avoid severe toxicities. clinicaltrials.gov Identifier: 00883623 (Eudract2008-001405-41).

Soft tissue sarcoma (STS) comprises a large variety of rare malignant tumors. Development of distant metastasis is frequent, even in patients undergoing initial curative surgery. Trabectedin, a tetrahydroisoquinoline alkaloid isolated from the Caribbean marine tunicate Ecteinascidia turbinata, was approved in 2007 for patients with advanced STS after failure of anthracyclines and ifosfamide, or for patients unsuited to receive these agents. In this study, we retrospectively analyzed 25 patients who had been treated with trabectedin at our institution between 2007 and 2010. The majority (72%) had been heavily pre-treated with ³2 previous lines of chemotherapy. Response assessed by conventional RECIST criteria was low, with only one patient achieving a partial remission (PR) and 10 stable disease (SD) after three cycles of treatment. However, median progression-free survival (PFS) and overall survival (OS) were significantly prolonged in this population compared to non-responders, with 7.7 months versus 2.1 months (p < 0.0001; HR 15.37, 95% CI 4.3 to 54.5) and 12.13 months versus 5.54 months (p = 0.0137; HR 3.7, 95% CI 1.3 to 10.5), respectively. PFS for all patients was 58% at three months and 37% at six months. Side effects, including neutropenia, elevation of liver transaminases/liver function tests, and nausea/vomiting, were usually mild and manageable. However, dose reductions due to side effects were necessary in five patients. We conclude that trabectedin is an effective and generally well tolerated treatment for STS even in a heavily pre-treated patient population.

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Background: We present the case of a 64-year-old patient who presented to his primary care physician with fatigue, worsening shortness of breath, abdominal discomfort and a rapidly growing abdominal girth, although he had lost 5 kg of weight within 3 months. He had a history of untreated hypertension, compensated renal insufficiency and COPD. Despite weight loss and fatigue, the patient did not experience any other constitutional symptoms such as fever, night sweats or loss of appetite. Investigations: Physical examination, blood tests, CT scan of the abdomen, MRI scan of the abdomen, fine needle biopsy, excisional biopsy, Video Capsule Endoscopy, histology, PET scan. Diagnosis: Inflammatory myofibroblastic tumor, chylous ascites, chyloperitoneum. Management: Systemic chemotherapy, total parenteral nutrition and octreotide therapy. Conclusion: We describe the case of a patient in whom two extremely rare phenomena are present in combination: the diagnosis of an inflammatory myofibroblastic tumor and chylous ascites. While the tumor could be stabilized by different regimens of chemotherapy, the chyloperitoneum was treated with parenteral nutrition and subcutaneous octreotide injections, which resulted in a significant reduction of the amount of chylous ascites drained during regular paracentesis.

Background: We used 2-deoxy-2-[18F] fluoro-D-glucose (FDG) positron emission tomography (PET) to evaluate the FDG uptake in patients with advanced and/or metastatic soft tissue sarcoma (STS) undergoing therapy with Ecteinascidin-743 (ET-743, Trabectedin, YondelisTM). Patients and Methods: The pilot study included nine patients with metastatic STS receiving a minimum of one cycle of treatment with trabectedin. Patients were examined using PET prior to onset of therapy and after completion of one or three cycles of trabectedin. Restaging according to Response Evaluation Criteria in Solid Tumours (RECIST) was performed in parallel using computed tomography (CT) and/or magnetic resonance imaging (MRI) and served for reference. Results: Clinical outcome of nine evaluable patients was as follows: one patient with partial remission (PR), three patients with stable disease (SD), and five patients with progressive disease (PD). A more than 40% decrease of the standardized uptake value (SUV) of sequential PET examination could be demonstrated for the responding patient (PR), whereas patients with SD or PD showed a stable SUV, but no increase in SUV. Conclusion: To our knowledge, this is the first small series of patients being treated with trabectedin and monitored using sequential PET imaging demonstrating SUV stabilization in nearly all monitored patients.

Steroid-refractory graft-versus-host disease is a life-threatening complication after allogeneic stem cell transplantation. Evidence is accumulating that steroid-refractory graft-versus-host disease is associated with endothelial distress. Endothelial cell homeostasis is regulated by nitric oxide, and serum nitrates are derived from nitric oxide synthase activity or dietary sources. In this retrospective study based on 417 patients allografted at our institution we investigated whether quantification of serum nitrates could predict steroid-refractory graft-versus-host disease. Elevated pre-transplant levels of serum nitrates (>26.5 μM) predicted steroid-refractory graft-versus-host disease (P=0.026) and non-relapse mortality (P=0.028), particularly in combination with high pre-transplant angiopoietin-2 levels (P=0.0007 and P=0.021, respectively). Multivariate analyses confirmed serum nitrates as independent predictors of steroid-refractory graft-versus-host disease and non-relapse mortality. Differences in serum nitrate levels did not correlate with serum levels of tumor necrosis factor or C-reactive protein or expression of inducible nitric oxide synthase in blood cells. Patients with high pre-transplant nitrate levels had significantly reduced rates of refractory graft-versus-host disease (P=0.031) when pravastatin was taken. In summary, patients at high risk of developing steroid-refractory graft-versus-host disease could be identified prior to transplantation by serum markers linked to endothelial cell function. Retrospectively, statin medication was associated with a reduced incidence of refractory graft-versus-host disease in this endothelial high-risk cohort.

In Europe, the combination of plerixafor + granulocyte colony-stimulating factor is approved for the mobilization of hematopoietic stem cells for autologous transplantation in patients with lymphoma and myeloma whose cells mobilize poorly. The purpose of this study was to further assess the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization in European patients with lymphoma or myeloma. In this multicenter, open label, single-arm study, patients received granulocyte colony-stimulating factor (10 μg/kg/day) subcutaneously for 4 days; on the evening of day 4 they were given plerixafor (0.24 mg/kg) subcutaneously. Patients underwent apheresis on day 5 after a morning dose of granulocyte colony-stimulating factor. The primary study objective was to confirm the safety of mobilization with plerixafor. Secondary objectives included assessment of efficacy (apheresis yield, time to engraftment). The combination of plerixafor + granulocyte colony-stimulating factor was used to mobilize hematopoietic stem cells in 118 patients (90 with myeloma, 25 with non-Hodgkin's lymphoma, 3 with Hodgkin's disease). Treatment-emergent plerixafor-related adverse events were reported in 24 patients. Most adverse events occurred within 1 hour after injection, were grade 1 or 2 in severity and included gastrointestinal disorders or injection-site reactions. The minimum cell yield (≥2×106 CD34+ cells/kg) was harvested in 98% of patients with myeloma and in 80% of those with non-Hodgkin's lymphoma in a median of one apheresis. The optimum cell dose (≥5×106 CD34+ cells/kg for non-Hodgkin's lymphoma or ≥6×106 CD34+ cells/kg for myeloma) was harvested in 89% of myeloma patients and 48% of non-Hodgkin's lymphoma patients. In this prospective, multicenter European study, mobilization with plerixafor + granulocyte colony-stimulating factor allowed the majority of patients with myeloma or non-Hodgkin's lymphoma to undergo transplantation with minimal toxicity, providing further data supporting the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization of hematopoietic stem cells in patients with non-Hodgkin's lymphoma or myeloma.

Steroid-refractory graft-versus-host disease causes significant morbidity and mortality after allogeneic stem cell transplantation. The pathomechanism of steroid resistance is currently not understood, but it has been suggested that endothelial cell dysfunction plays a role. Endothelial thrombomodulin was quantified along with histological markers of epithelial damage and cytotoxic T cells in colon biopsies from 51 allografted patients, and retrospectively correlated with response to steroids and survival. Loss of endothelial thrombomodulin was the strongest predictor of response to steroids (P=0.02) and nonrelapse mortality (P=0.01) in multivariate analyses adjusting for T-cell infiltrates, histological grading, vessel density, disease status, donor type, and conditioning therapy. Our data provide evidence that at disease onset, loss of endothelial thrombomodulin expression rather than excessive T-cell infiltration associates with steroid-refractory graft-versus-host disease and mortality. Prospective histological investigations are now warranted to improve diagnosis and prognostication of this core complication of stem cell transplantation.

Background. The therapeutic efficacy of allogeneic hemopoietic stem cell transplantation (HSCT) largely relies on the graft-versus-leukemia (GVL) effect. Uncontrolled graft-versus-host disease (GVHD) is a feared complication of HSCT. Regulatory T cells (Treg) are a subset of CD4+ T-helper cells believed to maintain tolerance after HSCT. It remains unclear whether low peripheral blood Treg have an impact on the risk for acute (aGVHD) and chronic GVHD (cGVHD). Methods. In this paper we enumerated the CD4+CD25highCD127low Treg in the peripheral blood of 84 patients after at least 150 days from HSCT and in 20 healthy age-matched controls. Results. Although similar mean lymphocyte counts were found in patients and controls, CD3+CD4+ T-cell counts were significantly lower in patients. Patients also had significantly lower Treg percentages among lymphocytes as compared to controls. Patients with cGVHD had even higher percentages of Treg if compared to patients without cGVHD. In multivariate analysis, Treg percentages were not an independent factor for cGVHD. Conclusions. This paper did not show a relation between deficient peripheral blood Treg and cGVHD, therefore cGVHD does not seem to occur as a result of peripheral Treg paucity.

Background Chromosomal abnormalities have been shown to play a major role in disease evolution of multiple myeloma. Specific changes in interphase cells can be detected by fluorescent in situ hybridization, which overcomes the problem of the lack of dividing cells required for conventional cytogenetics.Design and Methods We analyzed the prognostic value of 12 frequent chromosomal abnormalities detected by fluorescent in situ hybridization in a series of patients (n=315) with newly diagnosed, symptomatic multiple myeloma. All patients underwent frontline autologous stem cell transplantation according to the GMMG-HD3- or GMMG-HD4-trial protocols or analogous protocols.Results Univariate statistical analyses revealed that the presence of del(13q14), del(17p13), t(4;14), +1q21 and non-hyperdiploidy was associated with adverse progression-free and overall survival rates independently of the International Staging System (ISS) classification. Multivariate analyses showed that only t(4;14) and del(17p13) retained prognostic value for both progression-free and overall survival. According to the presence or absence of t(4;14) and del(17p13) and the patients’ International Staging System classification, the cohort could be stratified into three distinct groups: a group with a favorable prognosis [absence of t(4;14)/del(17p13) and ISS I], a group with a poor prognosis [presence of t(4;14)/del(17p13) and ISS II/III] and a group with an intermediate prognosis (all remaining patients). The probabilities of overall survival at 5 years decreased from 72% in the favorable prognostic group to 62% (hazard ratio 2.4; P=0.01) in the intermediate and 41% (hazard ratio 5.6; P

Background Research on mesenchymal stromal cells has created high expectations for a variety of therapeutic applications. Extensive propagation to yield enough mesenchymal stromal cells for therapy may result in replicative senescence and thus hamper long-term functionality in vivo. Highly variable proliferation rates of mesenchymal stromal cells in the course of long-term expansions under varying culture conditions may already indicate different propensity for cellular senescence. We hypothesized that senescence-associated regulated genes differ in mesenchymal stromal cells propagated under different culture conditions.Design and Methods Human bone marrow-derived mesenchymal stromal cells were cultured either by serial passaging or by a two-step protocol in three different growth conditions. Culture media were supplemented with either fetal bovine serum in varying concentrations or pooled human platelet lysate.Results All mesenchymal stromal cell preparations revealed significant gene expression changes upon long-term culture. Especially genes involved in cell differentiation, apoptosis and cell death were up-regulated, whereas genes involved in mitosis and proliferation were down-regulated. Furthermore, overlapping senescence-associated gene expression changes were found in all mesenchymal stromal cell preparations.Conclusions Long-term cell growth induced similar gene expression changes in mesenchymal stromal cells independently of isolation and expansion conditions. In advance of therapeutic application, this panel of genes might offer a feasible approach to assessing mesenchymal stromal cell quality with regard to the state of replicative senescence.

We evaluated the Serum Free Light Chain (FLC) test in a series of 133 untreated patients with systemic AL amyloidosis. The FLC test detected the monoclonal gammopathy in 87% compared with 92% for immunofixation of serum and urine in combination. However, both tests proved complementary. The FLC test was also a valuable tool in patients with advanced renal failure in spite of uninvolved light chain retention. Higher FLC levels were associated with higher bone marrow plasmocytosis, poorer Karnofsky index and heart involvement, and therefore reflected disease severity.

Background and Objectives Autologous stem cell transplantation (ASCT) is effective in mantle cell lymphoma (MCL). We investigated whether incorporation of rituximab into the high-dose regimen might further improve the results of ASCT in patients with MCL.Design and Methods In a prospective phase II study, patients with newly diagnosed MCL were treated with a sequential dose-escalating therapy comprising standard chemotherapy for remission induction, intensive ara-C-containing chemotherapy for mobilization of stem cells, and myeloablative therapy followed by ASCT. The myeloablative regimen consisted of total body irradiation and high-dose cyclophosphamide supplemented with two doses (375 mg/m3) of rituximab. Outcome parameters (toxicity, clinical and molecular response as assessed by allele-specific IGH real-time quantitative polymerase chain reaction (RQ-PCR), event-free survival, and overall survival) were compared with those of 34 historical controls treated identically but without rituximab.Results Thirty-four patients were accrued. Whereas engraftment, toxicity and clinical response were not different from those in controls, event-free survival was significantly increased with rituximab (not reached vs. 43 months; hazard ratio 0.38; p=0.036). This was associated with a trend for a superior molecular response rate in 11 study vs. 10 control patients with a marker available (73% vs. 30%, p=0.086) despite similar levels of lymphoma contamination of the stem cell inocula infused.Interpretation and Conclusions Incorporation of two standard doses of rituximab into the myeloablative regimen might improve outcome of upfront ASCT for MCL, allowing long-term disease control to an extent previously not reached in this disease.

The pyruvate transporter MPC1 (mitochondrial pyruvate carrier 1) acts as a tumour-suppressor, loss of which correlates with a pro-tumorigenic phenotype and poor survival in several tumour types. In high-grade serous ovarian cancers (HGSOC), patients display copy number loss of MPC1 in around 78% of cases and reduced MPC1 mRNA expression. To explore the metabolic effect of reduced expression, we demonstrate that depleting MPC1 in HGSOC cell lines drives expression of key proline biosynthetic genes; PYCR1, PYCR2 and PYCR3, and biosynthesis of proline. We show that altered proline metabolism underpins cancer cell proliferation, reactive oxygen species (ROS) production, and type I and type VI collagen formation in ovarian cancer cells. Furthermore, exploring The Cancer Genome Atlas, we discovered the PYCR3 isozyme to be highly expressed in a third of HGSOC patients, which was associated with more aggressive disease and diagnosis at a younger age. Taken together, our study highlights that targeting proline metabolism is a potential therapeutic avenue for the treatment of HGSOC.

Key Clinical Message Pembrolizumab is an immune checkpoint inhibitor with antitumor activity in other organ malignancies. We present this case —demonstrating multiple inflammatory adverse events associated with Pembrolizumab (in a single patient), in order to increase awareness and facilitate earlier identification of the wide‐ranging cutaneous side effects associated with immunotherapy.

We investigated various combination treatment regimens employing nilotinib with established chemotherapeutic agents (daunorubicin, mitoxantrone, etoposide and cytarabine) in imatinib-sensitive and -resistant BCR-ABL-positive cells. Mitoxantrone or cytarabine showed synergism (CI < 1) in combination with nilotinib in imatinib-sensitive LAMA84 cells, whereas in imatinib-resistant LAMA84-R cells synergistic effects could be assessed for daunorubicin, mitoxantrone and etoposide when combined with nilotinib. In both imatinib-sensitive and -resistant K562 cells daunorubicin, mitoxantrone and etoposide demonstrated synergism in combination with nilotinib. Moreover, both daunorubicin and mitoxantrone led to synergistic antiproliferative effects when combined with nilotinib in imatinib-resistant Ba/F3 cells carrying point mutations in the ABL TK domain (E255K, E255V and T315I). Annexin V/propidium iodide staining revealed a significant enhancement of nilotinib-induced apoptosis in imatinib-resistant Ba/F3T315I and LAMA84-R cells upon combination with daunorubicin and mitoxantrone, respectively. Our results demonstrate the efficacy of combination treatment regimens employing nilotinib and established chemotherapeutic agents in improving antileukemic effects in imatinib-sensitive and imatinib-resistant cells. This may be the foundation for further study on the potential of the applied combinations in a clinical setting. [ABSTRACT FROM AUTHOR]

SummaryThe mechanism that regulates self-renewal and differentiation of hematopoietic stem cells (HSC) is a central question in stem cell biology that might ultimately lead to reliable protocols for in vitro expansion of HSC. Cellular fate is governed by cell-cell interaction with the microenvironment in the bone marrow, the stem cell niche. Mesenchymal stromal cells (MSC) are precursors of the cellular components, and they secrete extracellular matrix proteins of the bone marrow stroma. Therefore, MSC feeder layer might provide a suitable in vitro model system for the stem cell niche. In vitro assays demonstrate that MSC maintain the stem cell function of HSC and that MSC from bone marrow have a higher hematopoiesis supportive activity than MSC from adipose tissue. Cocultivation with MSC might pave the way for expansion of long-term repopulating HSC, and various clinical trials indicate that co-transplantation of HSC and MSC might enhance engraftment. Thus, MSC are promising tools to elucidate the underlying mechanism of the cellular microenvironment. The large variety of preparative protocols for isolation and cultivation of MSC affects their stromal activity. Standardized isolation methods and molecular characterization of MSC are of utmost importance for reproducible isolation of hematopoiesis supportive stromal cells and for their potential clinical application.Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aims: Recent studies have shown that stem cell therapy may alleviate the detrimental effects of myocardial infarction. Yet, most of these reports observed only modest effects on cardiac function, suggesting that there still is need for improvement before widespread clinical use. One potential approach would be to increase migration of stem cells to the heart. We therefore tested whether local administration of stem cell factor (SCF) improves myocardial homing of intravenously infused lin-/c-kit+ stem cells after myocardial infarction. [ABSTRACT FROM PUBLISHER]

Background. High-dose chemotherapy followed by autologous blood stem cell transplantation induces remission of plasma cell dyscrasia in patients with AL amyloidosis. The impact of this treatment on the glomerular amyloid mass is still unknown. Methods. In the present study, the quantity of the renal amyloid mass before and more than 3 years after high-dose melphalan treatment and autologous blood stem cell transplantation was assessed in two patients. At the time of the second renal biopsy, both patients were in complete remission without detectable serum and urinary monocloncal IgA-λ and a normal percentage of plasma cells in the bone marrow. Results. In both patients with biopsy-proven AL amyloidosis, urinary protein excretion decreased from 7 g/24 h to <2 g/24 h more than 3 years after autologous blood stem cell transplantation. In contrast, glomerular amyloid deposits persisted, as shown in the second biopsy. Conclusion. Despite complete remission of the plasma cell dyscrasia and improvement of glomerular permeability, the amount of glomerular amyloid mass did not regress. [ABSTRACT FROM AUTHOR]

Our purpose was to assess the presence of centrosomal aberrations as measured by immunohistochemistry in primary invasive breast cancer and their association with established and proposed prognostic factors. Tissue sections of 103 primary invasive breast cancers were examined using centrosome-specific antibodies to pericentrin and γ-tubulin. At least 3 different tumor regions per case were examined to determine maximum centrosomal aberration levels, which represent the proportion of cells with abnormal centrosomes in the region with the highest percentage of cells with centrosomal aberrations. The χ2 test was performed to evaluate the association of maximum centrosomal aberration levels with patient age; tumor size; nodal status; nuclear grade; hormone receptor and Her2/neu expression; proportion of Ki67-, p53- and Bcl-2-positive tumor cells; DNA index; S-phase fraction; and proliferation index. With pericentrin immunohistochemistry, maximum centrosomal aberration levels >35% were detectable in 92 of the 103 breast carcinomas (89%). We found a highly significant correlation of maximum centrosomal aberration levels above 35% with axillary nodal tumor involvement (p < 0.0001) and the absence of hormone receptors (p < 0.0001). In addition, there was a borderline significant relationship with age <50 years (p = 0.050) and Her2/neu overexpression (p = 0.050). Among node-negative patients, maximum centrosomal aberration levels >35% were also associated with an increased DNA index (p = 0.006). In a subset of patients, additional staining of centrosomes with a monoclonal anti-γ-tubulin antibody essentially confirmed these results. In primary invasive breast cancer, centrosomal aberrations are associated with those factors predicting a more aggressive course of disease. This might indicate a fundamental role of centrosomal dysfunction in disease evolution, possibly as a result of chromosome missegregation during mitosis. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Myelodysplastic syndromes (MDS) are triggered by an aberrant hematopoietic stem cell (HSC). It is, however, unclear how this clone interferes with physiologic blood formation. In this study, we followed the hypothesis that the MDS clone impinges on feedback signals for self-renewal and differentiation and thereby suppresses normal hematopoiesis. Based on the theory that the MDS clone affects feedback signals for self-renewal and differentiation and hence suppresses normal hematopoiesis, we have developed a mathematical model to simulate different modifications in MDS-initiating cells and systemic feedback signals during disease development. These simulations revealed that the disease initiating cells must have higher self-renewal rates than normal HSCs to outcompete normal hematopoiesis. We assumed that self-renewal is the default pathway of stem and progenitor cells which is down-regulated by an increasing number of primitive cells in the bone marrow niche--including the premature MDS cells. Furthermore, the proliferative signal is up-regulated by cytopenia. Overall, our model is compatible with clinically observed MDS development, even though a single mutation scenario is unlikely for real disease progression which is usually associated with complex clonal hierarchy. For experimental validation of systemic feedback signals, we analyzed the impact of MDS patient derived serum on hematopoietic progenitor cells in vitro: in fact, MDS serum slightly increased proliferation, whereas maintenance of primitive phenotype was reduced. However, MDS serum did not significantly affect colony forming unit (CFU) frequencies indicating that regulation of self-renewal may involve local signals from the niche. Taken together, we suggest that initial mutations in MDS particularly favor aberrant high self-renewal rates. Accumulation of primitive MDS cells in the bone marrow then interferes with feedback signals for normal hematopoiesis--which then results in cytopenia.

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8(+) T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+) T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner.

The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.

Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.