Patrick Wuchter

A combination of granulocyte-colony-stimulating factor (G-CSF) and plerixafor mobilizes more primitive peripheral blood progenitor cells than G-CSF alone: results of a European phase II study

BACKGROUND AIMS Previous studies in xenograft models have shown that human peripheral blood progenitor cells (PBPC) mobilized with the CXCR4 antagonist plerixafor (AMD3100) have a higher bone marrow (BM) reconstitution potential than granulocyte-colony-stimulating factor (G-CSF)-mobilized PBPC. METHODS PBPC obtained during G-CSF-supported mobilization before and after a supplementary administration of AMD3100 from patients with multiple myeloma and non-Hodgkins lymphoma (n=15; phase II study) were investigated for co-expression of primitive and lineage-associated markers, their proliferative activity in vitro and repopulation potential after clinical transplantation. RESULTS A significant increase in primitive CD34+ CD38(-) cells was observed in intraindividual comparisons of all patients after administration of G-CSF+AMD3100 (peripheral blood: median 8-fold, range 2,4-fold - 39-fold) compared with G-CSF alone. Using a long-term culture-initiating cell assay, this increase was confirmed. After transplantation of G-CSF+AMD3100-mobilized PBPC, the time to leukocyte reconstitution > 1 x 10(3)/microL and platelet reconstitution > 2 x 10(4)/microL was 14 (10-19 days) and 13 days (10-15 days), respectively. A complete and stable hematologic reconstitution (platelets > 1.5 x 10(5)/microL) was observed in 91% of all patients within 35 days. CONCLUSIONS An additional application of AMD3100 to a standard G-CSF mobilization regimen leads to a significant increase in primitive PBPC with high repopulation capacity.

Aldehyde dehydrogenase activity among primary leukemia cells is associated with stem cell features and correlates with adverse clinical outcomes

OBJECTIVE Animal models have provided evidence for the existence of leukemia stem cells (LSC). However, prospective isolation of human LSC from patients with acute myeloid leukemia (AML), as well as the assessment of their clinical significance, has remained a major challenge. MATERIALS AND METHODS We have studied the functional characteristics of a subset of leukemia cells that expressed CD34 and high aldehyde dehydrogenase activity (ALDH(br)), which was freshly isolated from the mononuclear cells at the time of diagnosis from the marrow of 68 consecutive patients suffering from AML. RESULTS The percentage of ALDH(br) cells ranged from 0.01% to 16.0% with a median of 0.5%. Compared to their counterparts with low aldehyde dehydrogenase activity from the same individual patients, the ALDH(br) population showed a significantly higher affinity to human mesenchymal stromal cells (n=12; p<0.01), a more than twofold higher proportion of slow-dividing and quiescent cells (n=4; p<0.05), higher numbers of long-term culture-initiating cell colonies in vitro (n=25; p<0.01), and an enhanced engraftment in the nonobese diabetic/severe combined immunodeficient mouse model (n=3; p<0.05). Above all, we found that the frequency of ALDH(br) cells correlated significantly with diminished survival probability (p=0.025) and with adverse cytogenetic factors (p<0.05). CONCLUSION A small proportion of leukemia cells derived from the marrow of patients with AML were ALDH(br) and CD34(+). They demonstrated functional characteristics of LSC and high percentages of these cells among the leukemia cells correlated significantly with poor clinical outcome.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS The strategy for quality control testing depends on the products cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

N-cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells.

Specific cell-cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human-human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and beta-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.

Mesenchymal stromal cells (MSCs): science and f(r)iction

Due to their multi-lineage differentiation capacity, support of haematopoiesis, immunomodulation and secretion of proregenerative factors, mesenchymal stem/stromal cells (MSCs) are in the focus of intense research since decades. The literature is replete with reports on their potential in preclinical model systems. However, the heterogeneity of the primary cell population as starting material and the diverse protocols for isolation and cultivation are hampering progress in their clinical application. Consensus on common standards and harmonised isolation and characterisation protocols are important to ensure safety and efficacy. This review focuses on the recent scientific evidence of clinically relevant properties and on the speculative cardiomyogenic and hepatic differentiation potential of MSCs. Special emphasis is put on the importance of standardisation and harmonisation in clinical-scale manufacturing.

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

BACKGROUND AIMS Previous studies have demonstrated that the combination of granulocyte-colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34(+) hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets. METHODS We characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor. RESULTS The number of aldehyde dehydrogenase (ALDH)(bright) and CD34(+) cells was significantly higher after plerixafor treatment (1.2-5.0 and 1.5-6.0 times; both P < 0.01) and an enrichment of the very primitive CD34(+) CD38(-) and ALDH(bright) CD34(+) CD38(-) HSC subsets was detectable. Additionally, two distinct ALDH(+) subsets could be clearly distinguished. The small ALDH(high) subset showed a higher number of CD34(+) CD38(-) cells in contrast to the total ALDH(bright) subpopulation and probably represented a very primitive subpopulation of HSC. CONCLUSIONS A combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34(+) cells but was also able to increase the proportion of more primitive stem cell subsets.

Plerixafor is effective given either preemptively or as a rescue strategy in poor stem cell mobilizing patients with multiple myeloma

Harvest of more than one CD34+ stem cell transplant has become the standard, to ensure the option for a second autologous transplantation in patients with relapsed or progressive multiple myeloma (MM). Additional administration of the CXCR‐4 inhibitor plerixafor has been shown to increase the efficiency of CD34+ stem cell harvest. However, the algorithm when to apply plerixafor is still under debate.

Rescue stem cell mobilization with plerixafor economizes leukapheresis in patients with multiple myeloma

While extensive data demonstrated that plerixafor improves stem cell harvest in difficult‐to‐mobilize patients, economic concerns limit a broader application. We retrospectively assessed the effect of an early plerixafor rescue regimen for mobilization in patients with multiple myeloma. Patients were intended for high‐dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ABSCT) and therefore received cyclophosphamide‐based mobilization chemotherapy and consecutive stimulation with granulocyte colony‐stimulating factor (G‐CSF). Fifteen patients with poor stem cell harvest in the first leukapheresis session received plerixafor. Data were compared with a matched historic control group of 45 patients who also had a poor stem cell yield in the first apheresis session, but continued mobilization with G‐CSF alone. Patients in the plerixafor group collected significantly more CD34+ cells in total (median 4.9 vs. 3.7 [range 1.6–14.1 vs. 1.1–8.0] × 106 CD34+ cells /kg bw; P < 0.05), and also more CD34+ cells per leukapheresis procedure (P < 0.001). Consequently, they required a significantly lower number of leukapheresis procedures to achieve the collection goal (median 2.0 vs. 4.0 [range 2–3 vs. 2–9] procedures; P < 0.001). The efficiency of the collected stem cells in terms of hematologic engraftment after ABSCT was found to be equal in both groups. These data demonstrate that rescue mobilization with plerixafor triggered by a low stem cell yield in the first leukapheresis session is effective. Although the actual economic benefit may vary depending on the local leukapheresis costs, the median saving of two leukapheresis procedures offsets most of the expenses for the substance in this setting. An exemplary cost calculation is provided to illustrate this effect. J. Clin. Apheresis 29:299–304 2014.

Comparison between intermittent and continuous spectra optia leukapheresis systems for autologous peripheral blood stem cell collection.

Terumo BCT recently introduced a new system for mononuclear cell (MNC) collection that uses a Spectra Optia apheresis machine equipped with a redesigned disposable kit and software program (version 11.2). It allows for the continuous collection of MNCs, unlike the original Spectra Optia system (version 7.2), which included a chamber for two‐step cell separation. The aim of this study was to compare the two apheresis systems in regard to specific performance parameters. A retrospective data analysis of 150 patients who had undergone peripheral blood stem cell collection between March of 2014 and May of 2015 at our institution was performed. For the matched comparison, patients were divided into two groups by diagnosis and by previous forms of therapy received: a homogeneous group of patients with multiple myeloma (MM) that had received first line therapy (“MM” group, n = 88) and a heterogeneous group that included all of the other patients (“other” group, n = 62). No significant differences in CD34+ collection yields between both collection regimens were found (pMM = 0.19, pother = 0.74) in either group. Moreover, similar performance ratios (collected/predicted CD34+ cell number in %) were observed (pMM = 0.89, pother = 0.1). No relevant variations in platelet or hemoglobin loss were found between the two systems. We conclude that the new continuous Spectra Optia MNC system is equally efficient in collecting CD34+ cells and can be used without sacrificing collection efficiency levels when treating a broad variety of autologous patients. J. Clin. Apheresis 32:27–34, 2017.

The rarity of ALDH+ cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells; <1.9%; ALDH‐rare AML), whereas 24 patients had relatively numerous ALDH+ cells (≥1.9%; ALDH‐numerous AML). In patients with ALDH‐rare AML, normal HSC could be separated by their CD34+ALDH+ phenotype, whereas LSC were exclusively detected among CD34+ALDH− cells. For patients with ALDH‐numerous AML, the CD34+ALDH+ subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH+ cells from ALDH‐numerous AML were quiescent, refractory to ARA‐C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long‐term outcome were also characteristic for patients with ALDH‐numerous AML providing an additional risk‐stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.