Recruiting refugees and migrants as potential hematopoietic stem cell donors to serve patients of comparable ethnicities with rare human leucocyte antigen patterns - The BluStar.NRW project in North Western Germany.

Currently, approximately 19 million people with a migration background live in Germany. The majority of those descend from regions where the population has a genetically different distribution of HLA antigens when compared to the HLA frequencies usually found in North Western Europe. In case of severe haematological disorders of these individuals, allogeneic stem cell transplantation may be the treatment of choice. However, finding appropriate histocompatible hematopoietic stem cell donors continues to be a major challenge. If no matching sibling donors are available, there are only few suitable donors with a similar genetic background available in international blood stem cell donor registries. The "BluStar.NRW" project aimed to recruit new blood and hematopoietic stem cell donors with a migration background and to noticeably increase the number of suitable donors for patients within this group. Since December 2017, a total number of 9100 blood and stem cell donors with a migration background were recruited and typed for this project. HLA typing for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed by Next Generation Sequencing. We assessed the proportion of rare alleles according to HLA frequency tables, as defined by a frequency of <1:1000. The rare HLA allele frequencies according to HLA frequency tables of the BluStar.NRW cohort were compared with a matched control donor cohort: Rare HLA-A, -B, -C, -DRB1 and -DQB1 alleles occurred three times more frequent than in the control group, but rare HLA-DPB1 alleles occurred more frequently in the control cohort. This difference was highly significant for all HLA alleles (p < 0.0001 for HLA-A, -B, -C, -DRB1, -DPB1; p = 0.0002 for HLA-DQB1). In addition, the distribution of rare alleles differed between the two groups. To date, 29 work-ups were initiated, 12 PBSC, one BM and three DLI were collected so far out of the BluStar.NRW cohort. The apheresis probability is twofold higher (0.18% vs. 0.07%) compared to the control group which clearly shows a serious medical need. However, 13 work-ups were cancelled in the BluStar.NRW donor cohort which represents an almost twice as higher cancellation rate (45% vs. 25%). This single registry analysis with a large sample cohort clearly indicates that hematopoietic stem cell donors with a migration background represent an adequate donor pool to serve patients of comparable ethnicity.

The predictive and prognostic significance of cell-free DNA concentration in melanoma.

BACKGROUND: Melanoma is the leading cause of skin cancer-related deaths worldwide. While there have been significant improvements in the treatment of advanced melanoma in the past decade, biomarker development lagged behind. OBJECTIVES: The majority of liquid biopsy biomarkers rely on the analyses of oncogenic mutations; however, about 20% of melanoma patients are wild type. Therefore, validation of universal predictive and prognostic biomarkers is urgently needed. METHODS: We analysed plasma samples in a discovery cohort (n = 20) and expansion cohort (n = 166) of metastatic melanoma patients and healthy donors (n = 116). Total plasma circulating cell-free DNA (cfDNA) concentrations were measured on the Qubit® platform using assays for single-(ss) and double (ds)-stranded DNA, DNA spectrophotometry and RNase P qPCR. We explored the diagnostic, predictive and prognostic potential of cfDNA concentration by bio-statistical methods and established a cfDNA threshold for risk stratification. RESULTS: Our selected best method was Qubit® dsDNA assay which quantified higher plasma cfDNA concentrations in melanoma patients than in healthy controls (AUC 72%). Measurement of baseline cfDNA concentration revealed that high cfDNA was associated with presence of metastases and higher AJCC stage (P < 0.05). Furthermore, high baseline cfDNA was an indicator of shorter overall survival in patients with oncogenic mutations (HR 2.12, P = 0.0008), and in wild-type patients (HR 5.55, P < 0.0001). CONCLUSIONS: We provide evidence that total cfDNA can be used as a biomarker for melanoma irrespective of the tumour genotype and can provide information on tumour load, risk of progression and risk of death.

Elevated baseline serum PD-1 or PD-L1 predicts poor outcome of PD-1 inhibition therapy in metastatic melanoma.

BACKGROUND: Programmed cell death protein 1 (PD-1) checkpoint inhibition has recently advanced to one of the most effective treatment strategies in melanoma. Nevertheless, a considerable proportion of patients show upfront therapy resistance and baseline predictive biomarkers of treatment outcome are scarce. In this study we quantified PD-1 and programmed death-ligand 1 (PD-L1) in baseline sera from melanoma patients in relation to therapy response and survival. PATIENTS AND METHODS: Sera taken at therapy baseline from a total of 222 metastatic melanoma patients (two retrospectively selected monocentric discovery cohorts, n = 130; one prospectively collected multicentric validation cohort, n = 92) and from 38 healthy controls were analyzed for PD-1 and PD-L1 concentration by sandwich enzyme-linked immunosorbent assay. RESULTS: Melanoma patients showed higher serum concentrations of PD-1 (P = 0.0054) and PD-L1 (P < 0.0001) than healthy controls. Elevated serum PD-1 and PD-L1 levels at treatment baseline were associated with an impaired best overall response (BOR) to anti-PD-1 (P = 0.014, P = 0.041), but not to BRAF inhibition therapy. Baseline PD-1 and PD-L1 serum levels correlated with progression-free (PFS; P = 0.0081, P = 0.053) and overall survival (OS; P = 0.055, P = 0.0062) in patients who received anti-PD-1 therapy, but not in patients treated with BRAF inhibitors. By combining both markers, we obtained a strong discrimination between favorable and poor outcome of anti-PD-1 therapy, with elevated baseline serum levels of PD-1 and/or PD-L1 associated with an impaired BOR (P = 0.037), PFS (P = 0.048), and OS (P = 0.0098). This PD-1/PD-L1 combination serum biomarker was confirmed in an independent multicenter validation set of serum samples prospectively collected at baseline of PD-1 inhibition (BOR, P = 0.019; PFS, P = 0.038; OS, P = 0.022). Multivariable Cox regression demonstrated serum PD-1/PD-L1 as an independent predictor of PFS (P = 0.010) and OS (P = 0.003) in patients treated with PD-1 inhibitors. CONCLUSION: Our findings indicate PD-1 and PD-L1 as useful serum biomarkers to predict the outcome of PD-1 inhibition therapy in melanoma patients and to select patients for PD-1-based versus BRAF-based therapy strategies.

Early suppression of peripheral mononuclear blood cells in sepsis in response to stimulation with cytomegalovirus, OKT3, and pokeweed mitogen.

Critically ill patients are at risk for sepsis, and immunosuppressive mechanisms may prevail. Whether functional tests are helpful to detect immune alterations is largely unknown. Therefore, we tested the hypotheses that reactivity of peripheral blood mononuclear cells (PBMCs) to secrete interferon-γ (IFNγ) following stimulation in vitro is decreased in patients with early sepsis compared with postoperative patients. IFNγ secretion [enzyme-linked immunospot (ELISpot)] in response to stimulation with cytomegalovirus (CMV), pokeweed mitogen (PWM), muromonab-anti-CD3 (OKT3), and human leukocyte antigen (HLA)-DRA-mRNA expression and serum cytokine concentrations were repeatedly [days 1, 3, 5, and 7 after intensive care unit (ICU) admission] determined in patients with sepsis (n = 7) and patients undergoing major abdominal surgery (radical prostatectomy, cystectomy, n = 10). In a second cohort, HLA-DRA expression was assessed in 80 patients with sepsis, 30 postoperative patients, and 44 healthy volunteers (German clinical trials database no. 00007694). In patients with sepsis, IFNγ secretion (ELISpot) was decreased compared with controls after stimulation with CMV (P = 0.01), OKT3 (P = 0.02), and PWM (P = 0.02 on day 5), whereas unstimulated IFNγ secretion did not differ. HLA-DRA expression was also significantly decreased in patients with sepsis at all time points (P = 0.004) compared with postoperative surgical patients, a finding confirmed in the larger cohort. Reactivity of PBMCs to stimulation with CMV, PWM, and OKT3 as well as HLA-DRA expression was already decreased upon ICU admission in patients with sepsis when compared with postoperative controls, suggesting early depression of acquired immunity. ELISpot assays may help to clinically characterize the time course of immunocompetence in patients with sepsis.NEW & NOTEWORTHY We observed suppression of reactivity to stimulation with cytomegalovirus, muromonab-anti-CD3, and pokeweed mitogen in mononuclear blood cells of patients with early sepsis when compared with postoperative controls. Thus, there is early depression of acquired immunity in sepsis. Enzyme-linked immunospot assays may help to characterize immunocompetence in patients with sepsis.

Primary central nervous system vasculitis and its mimicking diseases - clinical features, outcome, comorbidities and diagnostic results - A case control study.

OBJECTIVES: To compare clinical features and outcome, imaging characteristics, biopsy results and laboratory findings in a cohort of 69 patients with suspected or diagnosed primary central nervous system vasculitis (PCNSV) in adults; to identify risk factors and predictive features for PCNSV. PATIENTS AND METHODS: We performed a case-control-study including 69 patients referred with suspected PCNSV from whom 25 were confirmed by predetermined diagnostic criteria based on biopsy (72%) or angiography (28%). Forty-four patients turned out to have 15 distinct other diagnoses. Clinical and diagnostic data were compared between PCNSV and Non-PCNSV cohorts. RESULTS: Clinical presentation was not able to discriminate between PCNSV and its differential diagnoses. However, a worse clinical outcome was associated with PCNSV (p=0.005). Biopsy (p=0.004), contrast enhancement (p=0.000) or tumour-like mass lesion (p=0.008) in magnetic resonance imaging (MRI), intrathecal IgG increase (p=0.020), normal Duplex findings of cerebral arteries (p=0.022) and conventional angiography (p 0.010) were able to distinguish between the two cohorts. CONCLUSION: In a cohort of 69 patients with suspected PCNSV, a large number (64%) was misdiagnosed and partly received treatment, since mimicking diseases are very difficult to discriminate. Clinical presentation at manifestation does not help to differentiate PCNSV from its mimicking diseases. MRI and cerebrospinal fluid analysis are unlikely to be normal in PCNSV, though unspecific if pathological. Cerebral angiography and biopsy must complement other diagnostics when establishing the diagnosis in order to avoid misdiagnosis and mistreatment. CLINICAL TRIAL REGISTRATION: German clinical trials register: http://drks-neu.uniklinik-freiburg.de/drks_web/, Unique identifier: DRKS00005347.

Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions.

UNLABELLED: Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. INTRODUCTION: Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. METHODS: cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, ß-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. RESULTS: In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. CONCLUSIONS: The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

Development of patient-specific hematopoietic stem and progenitor cell grafts from pluripotent stem cells, in vitro.

Pluripotent stem cells hold great promise for future applications in many areas of regenerative medicine. Their defining property of differentiation towards any of the three germ layers and all derivatives thereof, including somatic stem cells, explains the special interest of the biomedical community in this cell type. In this review, we focus on the current state of directed differentiation of pluripotent stem cells towards hematopoietic stem cells (HSCs). HSCs are especially interesting because they are the longest known and, thus, most intensively investigated somatic stem cells. They were the first stem cells successfully used for regenerative purposes in clinical human medicine, namely in bone marrow transplantation, and also the first stem cells to be genetically altered for the first successful gene therapy trial in humans. However, because of the technical difficulties associated with this rare type of cell, such as the current incapability of prospective isolation, in vitro expansion and gene repair by homologous recombination, there is great interest in using pluripotent stem cells, such as Embryonic Stem (ES-) cells, as a source for generating and genetically altering HSCs, ex vivo. This has been hampered by ethical concerns associated with the use of human ES-cells. However, since Shinya Yamanaka´s successful attempts to reprogram somatic cells of mice and men to an ES-cell like state, so-called induced pluripotent stem (iPS) cells, this field of research has experienced a huge boost. In this brief review, we will reflect on the status quo of directed hematopoietic differentiation of human and mouse pluripotent stem cells.

Regenerative therapies in neonatology: clinical perspectives.

Regenerative therapy based on stem cells is applied as standard therapy in pediatric oncology. Furthermore, they are frequently used to treat immunodeficiency disorders of infants. For severe neonatal diseases, e. g. hypoxic-ischemic encephalopathy in term neonates or bronchopulmonary dysplasia in preterm infants, animal models have been established. According to some first preclinical results stem cell administration appears as a promising tool to improve the clinical outcome in high-risk infants. Provided the benefit of regenerative therapies can further be evaluated in appropriate preclinical neonate models, carefully controlled clinical trials to assess the significance of regenerative therapies, such as autologous stem cell administration, are indicated.

Lymphocyte function following radioiodine therapy in patients with thyroid carcinoma.

AIM: Since the nuclear disaster in Fukushima has raised great concern about the danger of radioactivity, we here addressed the question if the therapeutic use of iodine 131, the most frequently applied radionuclide, was harmful to immune function in patients. It was our aim to define for the first time in a clinical setting how radioiodine therapy alters anti-microbial immune responses. PATIENTS, METHODS: In 21 patients with thyroid carcinoma anti-microbial lymphocyte responses were assessed by lymphocyte transformation test and ELISpot - measuring lymphocyte proliferation and on a single cell level production of pro- and anti-inflammatory cytokines (interferon-γ and interleukin-10) - prior to therapy, at day 1 and day 7 post therapy. RESULTS: Proliferative lymphocyte responses and interferon-γ production after in vitro stimulation with microbial antigens were significantly (p < 0.05) increased at day 1 vs. pre therapy, and returned to pre therapy levels at day 7. On the contrary, at day 1 interleukin-10 production was significantly (p < 0.05) reduced. Thus, we observed a short-term increase in pro-inflammatory immune responses. However, T lymphocyte responses were in the range of healthy controls at all three time points. CONCLUSION: Thyroid carcinoma patients receiving radioiodine therapy do not display any sign of immunosuppression.

The prognostic significance of soluble NKG2D ligands in B-cell chronic lymphocytic leukemia.

Soluble or membrane-anchored ligands of NKG2D and their receptor have a critical role in the elimination of tumor cells and disease progression. Plasma samples of 98 patients with B-cell chronic lymphocytic leukemia (CLL) were analyzed with specific ELISA systems for soluble major histocompatibility complex class I-related chains (sMICA and sMICB) and UL-16-binding proteins (ULBP1, 2, and 3). The flow cytometric analysis of MICA on CLL cells and natural killer group 2 member D (NKG2D) receptors on NK cells was performed after thawing of frozen peripheral blood lymphocytes of CLL patients (N=51). Levels of sMICA, sMICB, and sULBP2 were significantly increased (P<0.001) compared with 48 controls, whereas sULBP1 3 were not detectable in patients and controls. Levels of sMICA>990 pg/ml (P=0.014), sMICB>200 pg/ml (P=0.0001), and sULBP2>105 pg/ml (P<0.0001) were associated with poor treatment-free survival (TFS). Neither MICA nor NKG2D expression could be related to clinical parameters. In multivariate analysis Binet stage (P=0.002), sULBP2 (P=0.002) and ZAP-70 (P=0.002) were independent predictive factors for TFS. In patients with Binet stage A, sULBP2 levels>105 pg/ml were strongly associated (P=0.0025) with poor TFS. Our data show that soluble but not membrane-anchored NKG2D ligands or receptors are of prognostic significance in CLL. Moreover, sULBP2 seems to be useful to identify early-stage patients with risk of disease progression.

DQB1*0323 is the result of a gene conversion event between a DQB1*06 and a DQB1*030303 allele.

A novel human leukocyte antigen-DQB1 allele, DQB1*0323, was identified in a volunteer hematopoietic stem cell donor. DQB1*0323 differs from the closely related allele DQB1*030303 in five nucleotide positions.

Identification of a novel HLA-DQB1 allele, HLA-DQB1*0632.

A novel human leukocyte antigen-DQ allele, DQB1*0632, was identified in a 68-year-old bone marrow transplantation candidate. DQB1*0632 differs from DQB1*0603 by one nucleotide change in exon 2 resulting in the amino acid exchange Gly --> Arg.

The nature of diversity of HLA-DRB1 exon 3.

Exon 3 of DRB1 is known to be polymorphic, but thought to be conserved within allelic groups. This implies that exon 3 polymorphisms would not need to be considered in evolutionary studies or clinical settings when assessing immunogenicity of allelic mismatches in stem cell transplantation. To further assess this, we determined the sequences of DRB1 exon 3 by hemizygote amplification and direct sequencing on 55 selected DNA samples containing 42 DRB1 alleles for which no exon 3 sequence data were previously available. The data confirmed the high degree of overall sequence conservation. The DRB4- and DRB5-associated alleles were completely conserved within their DRB1 groups. However, it could be shown that exon 3 is more diverse than previously expected. Multiple allelic differences within each group of DRB3-associated DRB1 alleles were found, without identifying unique group-related sequence motifs differentiating between these groups. For DRB1*1402 and DRB1*1406, it could be shown that they originated from DRB1*0302. In several samples previously typed as DRB1*1401, a novel DRB1 allele was identified: DRB1*1454. Thus, from a clinical viewpoint, the availability of exon 3 sequence information may be useful for optimizing typing as well as matching strategies. Additionally, it will allow for more detailed evolutionary studies, further elucidating the origin of alleles and the mechanisms driving sequence diversification.

The variation of the novel allele HLA-B*0739 suggests low alloreactivity when mismatched with HLA-B*0702.

We here report the identification of a new HLA-B*07 allele in a male Caucasian. The new allele was initially typed as B*0713 by sequence-specific primed PCR. Because of the infrequence of that allele, a sequencing-based typing was performed to confirm that result. This yielded the detection of the novel allele. It is closest to B*070201, while it differs from B*0713 in 12 positions in exon 2. Compared to B*070201, the new variant is characterized by a non-synonymous nucleotide exchange (C-->T) at nucleotide position 118 of exon 2. Previously, this was considered a constant position, suggesting that it is likely to be caused by a single-point mutation. It results in the amino acid exchange Ala-->Val at position 40 of the mature polypeptide. As this position is located in an outer loop of the HLA molecule, it is highly unlikely to affect peptide binding or T-cell receptor interaction. Thus, the newly found allele should have a low alloreactive potential in case of a mismatch to the most common HLA-B allele B*0702.

Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons.

Efficient transduction of hematopoietic stem cells is a prerequisite for successful hematopoietic stem cell gene therapy. Oncoretroviral vectors are the most widely used vectors for hematopoietic gene therapy studies. However, these vectors require cell division, and thus efficient transduction of quiescent stem cells has been difficult to achieve. Lentiviral vectors can transduce non-dividing cells and therefore may be more efficient in transducing quiescent hematopoietic stem cells. We have used a competitive repopulation assay in the baboon to compare transduction of hematopoietic repopulating cells by lentiviral and oncoretroviral vectors. Baboon CD34-enriched marrow cells were transduced in the presence or absence of multiple hematopoietic growth factors using a short, 2-day, transduction protocol. Here, we show that efficient lentiviral transduction of hematopoietic repopulating cells was only achieved when cells were transduced in the presence of multiple growth factors. Using these conditions, up to 8.6% of hematopoietic repopulating cells were genetically modified by the lentiviral vector more than 1 year after transplant. Interestingly, the number of lentivirally marked cells increased over time in three of four animals. In conclusion, these results suggest that lentiviral vectors are able to tranduce multilineage hematopoietic stem cells, and thus, may provide an alternative vector system for clinical stem cell gene therapy applications.

Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.

Previous studies have shown that the choice of envelope protein (pseudotype) can have a significant effect on the efficiency of retroviral gene transfer into hematopoietic stem cells. This study used a competitive repopulation assay in the dog model to evaluate oncoretroviral vectors carrying the envelope protein of the endogenous feline virus, RD114. CD34-enriched marrow cells were divided into equal aliquots and transduced with vectors produced by the RD114-pseudotype packaging cells FLYRD (LgGLSN and LNX) or by the gibbon ape leukemia virus (GALV)-pseudotype packaging cells PG13 (LNY). A total of 5 dogs were studied. One dog died because of infection before sustained engraftment could be achieved, and monitoring was discontinued after 9 months in another animal that had very low overall gene-marking levels. The 3 remaining animals are alive with follow-ups at 11, 22, and 23 months. Analyses of gene marking frequencies in peripheral blood and marrow by polymerase chain reaction revealed no significant differences between the RD114 and GALV-pseudotype vectors. The LgGLSN vector also contained the enhanced green fluorescent protein (GFP), enabling us to monitor proviral expression by flow cytometry. Up to 10% of peripheral blood cells expressed GFP shortly after transplantation and approximately 6% after the longest follow-up of 23 months. Flow cytometric analysis of hematopoietic subpopulations showed that most of the GFP-expressing cells were granulocytes, although GFP-positive lymphocytes and monocytes were also detected. In summary, these results show that RD114-pseudotype oncoretroviral vectors are able to transduce hematopoietic long-term repopulating cells and, thus, may be useful for human stem cell gene therapy.