Thomas Fietz

Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells.

We compared UCB mononuclear cells (MNC) with CD34+ selected cells in a serum-free static culture system. Cell number proliferation of MNCs was inferior to CD34+ selected cells. MNCs, however, showed a substantial increase from 0.94% CD34+ cells on day 0 to 5.8% on day 7, whereas in the CD34+selected samples the CD34+ cell content declined continously from 62.2% on day 0 to 27.7% on day 7. The number of CFU-GM increased during culture of both cell fractions. Here, only the MNCs showed a substantial increase in clonogenicity on day 7 and day 14 to 11.1- and 4.1-fold input, respectively. This expansion of the CD34+ progenitor cell pool in the MNCs fraction was at least in part attributable to T cells, since the physical abrogation of T cells blocked this effect. Refeeding and reseeding of cells on day 7 had stimulating effects especially on the CD34+ cells, where cell number proliferation increased from 16.3-fold without to 58.1-fold on day 14. Also, we could find sporadic chromosomal aberrations in four of 100 metaphases examined after 7–20 days of ex vivo expansion. The significance of this observation needs to be clarified in a larger series.

Intramedullary abscess in a patient with disseminated Scedosporium apiospermum infection

A 35-year-old man was admitted with a relapsed acute lymphoblastic leukaemia (ALL). The initial diagnosis had been made 4 years earlier and at that time a proven invasive pulmonary aspergillosis had been successfully treated with voriconazole. Following initial chemotherapy, the patient entered complete remission. Following relapse, re-induction chemotherapy was given. During this chemotherapy, disseminated bluish-red cutaneous lesions developed over the entire trunk and head, despite oral itraconazole prophylaxis. The lesions initially had a clear vesicle, but subsequently developed central necrosis (top). A skin biopsy showed filamentous fungal elements (bottom left). Despite amphotericin B, liposomal amphotericin B, allogeneic granulocyte transfusions and, finally, voriconazole therapy, the fungal infection progressed with a microbiologically confirmed intraocular mycosis, orbital cellulitis and an intramedullary abscess (bottom right, arrow). Microbiological testing ultimately detected Scedosporium apiospermum. Despite sensitivity to voriconazole (and resistance to itraconazole, fluconazole, flucytosine and amphotericin B), confirmed by in vitro testing, and recovery from neutropenia, the patient developed multiple brain abscesses and died of progressive fungal infection. Scedosporium is a rare filamentous fungus subdivided into two species: S. prolificans and S. apiospermum. Because of its broad resistance to antifungal agents, infections in immunocompromised patients are often fatal.

Ex vivo expansion of human umbilical cord blood does not lead to co-expansion of contaminating maternal mononuclear cells

One of the main limiting factors for increased use of human umbilical cord blood (UCB) in adult allogeneic transplantation is the small number of progenitor cells that can be collected and infused. Ex vivo expansion of UCB might help to overcome this limitation. Whether an expansion of UCB cells will also lead to co-expansion of contaminating maternal cells, and thus may alter graft characteristics and lead to an increased incidence of GVHD, has not been looked at so far. We initiated cultures with UCB mononuclear cells (MNC) in a standard medium containing stem cell factor (SCF), flt-3L, Il-3, IL-6, EPO and G-CSF. To address the question of contaminating maternal cells we performed interphase FISH analysis of the X and Y chromosome simultaneously. Male (XY) cord blood samples were investigated for maternal (XX) cells at day 0 and at several time points during culture. We could not detect maternal cells in any of the nine samples studied when cultures were started at day 0. Culturing did not expand previously undetected maternal cells into a range that could be seen with FISH technology, as all samples remained negative for maternal cells throughout culture periods of 14 days. We then artificially contaminated male UCB with maternal mononuclear cells at concentrations of 5 and 15% at day 0. After 14 days, maternal MNC were still detectable, but the percentage was reduced to 1.7% and 6%, respectively. During culturing of CD34+-selected UCB the content of maternal cells also declined from a mean of 1.6% after contamination to 0.4% on day 7. Taken together we could show that maternal cells co-cultured with UCB do not co-expand and thus do not interfere with ex vivo expansion of UCB for adult allogeneic transplantation.

CD34 selected alloPBSCT and adoptive immunotherapy.

To circumvent aGVHD in the early phase after allogeneic stem cell transplantation but to provide GVL activity later on, we performed alloPBSCT with CD34+ selected grafts followed by delayed add-back of CD3+ T cells. Ten consecutive patients having an HLA-identical sibling donor were enrolled on to this trial. Four patients were in first CR of high-risk ALL, another four in first CR of AML, one was in second myeloid blast crisis of CML, and one was in PR of relapsed NHL. Conditioning consisted of 2 × 60 mg/kg CY plus 12 Gy TBI. G-CSF (Filgrastim) mobilized peripheral cells were CD34+selected using the Isolex 300i system in nine patients and the CliniMacs system in one. Median CD34+ purity was 86%. A median of 2.8 × 106/kg CD34+ cells were transplanted. The number of CD3+ cells in the allografts was 5.7 × 104/kg (median) after Isolex 300i, and 0.2 × 104/kg after CliniMacs. All patients received G-CSF (Filgrastim) and engrafted rapidly. Standard-dose CsA was administered, and until day +60 no aGVHD occurred. At that time point, seven patients received 2 × 106/kg CD3+ cells while CsA had been tapered to 50% of the starting dose. One of these patients died after a second T cell boost given on day +90 without concomitant immunosuppression due to grade IV intestinal aGVHD. Three others developed cutaneous cGVHD. Taken together, T cell depletion by CD34+ selection does not impair rapid engraftment in the HLA-identical sibling donor setting. Using standard-dose CsA the risk for acute GVHD seems to be minimized. Add-back of 2 × 106/kg CD3+ cells on day +60 with CsA protection is feasible. However, whether this is the optimal time point and number of T cells remain to be further elucidated. Bone Marrow Transplantation (2000) 25, Suppl. 2, S2–S5.

MDR-1 Expression and Deletions of Chromosomes 7 and 5(Q) Separately Indicate Adverse Prognosis in AML

In order to assess any correlation between MDR-1 expression and chromosomal aberrations, and to define their impact on clinical outcome in newly diagnosed AML pts, we investigated bone marrow and peripheral blood samples of 49 consecutive pts admitted to our hospital. Monosomy 7, trisomy 8 and 5q-were evaluated by means of interphase fluorescence in situ hybridization (FISH). Monosomy 7 was present in 6 pts, trisomy 8 in 5 pts, and 5q-in 6 pts. More than one aberration was seen in 7 pts. Chromosomal aberrations were mostly found in older pts (12/14 > 60 years; p=0.03) and in pts with CD34 positive leukemic blasts (13/14 coexpressed CD34; p=0.0004). In 25 pts also standard G-banding analysis was performed leading to concordant results regarding chromosomes 7, 8 and 5. Flow cytometry identified MDR-1 positivity (MDR+) in 16 pts. MDR-1 expression appeared to be a characteristic feature in CD34+ AML (12/16 were CD34+ and MDR+ pts; p=0.013). No correlation, however, was found between chromosomal aberrations and MDR-1 expression. Pts with aberrations of either chromosomes 7, 8 or 5 detected by FISH (FISH+) were predominantly resistant to induction therapy (6/8 pts, p=0.004). A lower rate of complete remission (CR) was also seen in pts with MDR-1 expression (p=0.006). MDR+/FISH+ pts (n=3) were all refractory to remission induction, while all MDR-/FISH-pts (n=19) achieved CR (p=0.0006). MDR-1+ as well as pts with aberrations of chromosomes 7, and 5(q) showed a significantly decreased probability of overall survival. In conclusion, MDR-1 expression as well as abnormalities of chromosomes 7, and 5(q) predict poor clinical outcome in AML. The identification of these prognostic factors provides useful information for risk adapted treatment strategies.

Favourable outcomes of poor prognosis diffuse large B‐cell lymphoma patients treated with dose‐dense Rituximab, high‐dose Methotrexate and six cycles of CHOP‐14 compared to first‐line autologous transplantation

The optimal therapeutic approach for young diffuse large B‐cell lymphoma (DLBCL) patients with high‐intermediate and high‐risk age‐adjusted international prognostic index (aaIPI) remains unknown. Hereby we report a 10‐year single‐centre study of 63 consecutively treated patients. To optimize outcome, two approaches were carried out: Cohort 1 patients received four cycles R‐CHOP‐21 (rituximab, cyclophosphamide, daunorubicin, vincristine, prednisolone over 21 days) followed by first‐line high‐dose chemotherapy with autologous stem‐cell support (HDCT‐ASCT), resulting in 2‐year progression‐free (PFS) and overall survival (OS) of 60·6% and 67·9%. 39·4% of those patients were not transplanted upfront, mainly due to early progressive disease (24·2%). Cohort 2 patients received an early intensified protocol of six cycles of CHOP‐14 (cyclophosphamide, daunorubicin, vincristine, prednisolone over 14 days) with dose‐dense rituximab and high‐dose methotrexate resulting in promising overall response‐ (93·3%) and complete remission (90%) rates and sustained survival (2‐year PFS and OS: 93·3%). In an intention‐to‐treat analysis, 2‐year PFS (60·6% vs. 93·3%, hazard ratio [HR] 7·2, P = 0·009) and OS (69·7% vs. 93·3%, HR 4·95, P = 0·038) differed significantly, in favour of the early intensified protocol (Cohort 2). In a multivariate Cox‐regression model, PFS (HR 8·12, 95% confidence interval [CI] 1·83–35·9, P = 0·006) and OS (HR 5·86, 95% CI 1·28–26·8, P = 0·02) remained superior for Cohort 2 when adjusted for aaIPI3 as the most important prognostic factor. Survival of young poor‐prognosis DLBCL patients appears superior after early therapy intensification.

Simultaneous Double-Purging of Breast Cancer Cells from Leukapheresis Products by Immunomagnetic CD34+ Cell Enrichment and Tumor Cell Depletion

During the last few years breast cancer has become an important field for peripheral blood progenitor cell 3transplantation (PBPCT) following high dose (HD) chemotherapy [2]. The high prognostic value of bone marrow micrometastasis and the mobilization of malignant cells into the peripheral blood following priming for PBPC collection are well known [5,7,18]. Although the negative influence of tumor cells contaminating autografts on the clinical outcome after stem cell retransfusion is still not proven by prospective clinical studies, the reduction of residual tumor cells is a main target of leukapheresis product (LP) processing. Immunomagnetic CD34+ cell enrichment (Baxter Isolex 300i system) is a widely applied purging procedure (+ selection) [13]. An additional second immunomagnetic purging (− selection) step can increase the tumor cell depletion [3,14]. We applied a new simultaneous +/- immunomagnetic purging method in an experimental setting and in clinical samples. Additionally, we compared the results with our own clinical data from patients who underwent stem cell rescue with PBPC samples purged by CD34+ selection only or toxic lipid (ET-18-OCH3) incubation.