The impact of allogeneic stem cell transplantation on the natural course of poor-risk chronic lymphocytic leukemia as defined by the EBMT consensus criteria: a retrospective donor versus no donor comparison.

BACKGROUND: In a single-center retrospective donor versus no-donor comparison, we investigated if allogeneic stem cell transplantation (alloSCT) can improve the dismal course of poor-risk chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: All patients with CLL who were referred for evaluation of alloSCT within a 7-year time frame and had a donor search indication according to the EBMT criteria or because of Richter's transformation were included. Patients for whom a matched donor could be found within 3 months (matches) were compared with patients without such a donor (controls). Primary end point was overall survival measured from the 3-month landmark after search initiation. RESULTS: Of 105 patients with donor search, 97 (matches 83; controls 14) were assessable at the 3-month landmark. Matches and controls were comparable for age, gender, time from diagnosis, number of previous regimens, and remission status. Disregarding if alloSCT was actually carried out or not, survival from the 3-month landmark was significantly better in matches versus controls [hazard ratio 0.38, 95% confidence interval (CI) 0.17-0.85; P = 0.014]. The survival benefit of matches remained significant on multivariate analysis. CONCLUSION: This study provides first comparative evidence that alloSCT may have the potential to improve the natural course of poor-risk CLL as defined by the EBMT criteria.

[Mobile geriatric rehabilitation in functionally severely impaired patients. Investigations on effectiveness]. / Mobile geriatrische Rehabilitation bei funktionell schwer beeinträchtigten Patienten. Untersuchungen zur Effektivität.

INTRODUCTION: Mobile geriatric rehabilitation is an outpatient rehabilitative treatment in which a multidisciplinary team treats elderly patients at home. This kind of treatment has been performed in rare cases in Germany but there are no data available on the effectiveness in patients with severe cognitive and functional impairment. MATERIAL AND METHODS: In a retrospective study design the data of all patients who had participated in mobile geriatric rehabilitation between 1 September 2009 and 23 May 2011 were evaluated. Before treatment a comprehensive geriatric assessment was performed and after treatment an assessment of mobility and activities of daily living (ADL). After 6 months a random sample of 20 patients were contacted by telephone to check the ADL. RESULTS: A total of 87 patients were treated between 1 September 2009 and 23 May 2011. The median age was 83 years and 56 % of the patients lived in nursing homes. Only 24 % of the patients had MMSE scores > 23 points, 77 patients completed the treatment with an assessment and in these patients the ADL could be improved significantly (Barthel index at the beginning 36.2 points and on completion 50.9 points, p < 0.001). The Barthel index 6 months after treatment was only 1.25 points lower compared to the assessment at the end of the treatment. Significant improvement after therapy could also be demonstrated in the mobility assessment (timed up and go test, Tinetti mobility score and Esslinger transfer scale). CONCLUSION: The results of this non-randomized and non-blinded trial indicated the efficacy of mobile geriatric rehabilitation. In functionally and cognitively impaired elderly patients ADL and mobility can be improved. These effects seem to persist for at least for 6 months.

Treatment of primary mediastinal large B cell lymphoma with an alternating chemotherapy regimen based on high-dose methotrexate.

Primary mediastinal large B cell lymphomas (MLCL) differ from other diffuse large cell lymphomas, leading to a description as a separate entity in the current World Health Organization classification. Dose intensification improves long-term results, but no standard therapy has been established so far. We investigated the use of a high-dose methotrexate-based alternating chemotherapy regimen (B-ALL protocol of the German ALL study group) followed by consolidative mediastinal radiotherapy first as a single-center trial, then later as a prospective multicenter trial in 44 patients with a median age of 33 years. Response rates exceeded 90% with an overall survival rate of 80% in the single-center group (8.6 years median follow-up) and 82% in the multicenter group (2.5 years follow-up).Short-term toxicity was manageable, but required hospitalization: the rates of grade 3 or 4 toxicity were 20% (for mucositis), 42% (for neutropenia), 29% (for thrombocytopenia), and 9% (for neutropenic fever). No relapse occurred more than 2 years after diagnosis and initiation of treatment, but unfortunately, no patient with overt progression or relapse within these 2 years could be salvaged. Future directions in the treatment of MLCL will not focus on further dose intensification, but rather on the incorporation of (radio)immunotherapy as a therapeutic tool and gene expression profiling as well as positron emission tomography-computed tomography as stratifying tools.

Evidence for the presence of xenopsin-related peptide(s) in the gastric mucosa of mammals.

Using immunohistochemistry and radioimmunoassay, substance(s) related to the amphibian octapeptide xenopsin (XP) were demonstrated in the gastric mucosa of humans and dogs. Immunohistochemistry localized XP-immunoreactive epithelial cells in the gastric antral mucosa. The reaction was abolished by preabsorption of the antiserum with XP but not by neurotensin or other peptides. Immunoreactive XP (iXP) was found by radioimmunoassay in extracts of both the antrum and body of the stomach prepared with acid/acetone or acetic acid. A study of its distribution in the dog indicated that the level of iXP was highest in the stomach, lower in the pancreas and duodenum, and not measurable in the jejunoileum and colon. Gel chromatography on Sephadex G-25 indicated the presence of at least two forms of iXP, one larger and the other about the same size as XP. Reverse-phase high pressure liquid chromatography on mu-Bondapak C-18 yielded several peaks of iXP, one of which eluted at the position of synthetic XP. The results of immunochemical analyses using four different antisera towards XP were consistent with structures for canine iXPs that were closely related to XP only in their C-terminal regions. These results suggest that mammalian counterparts to amphibian XP reside within endocrine cells of the gastric mucosa. It seems possible that these peptides function as gastrointestinal signals.

Reduced-toxicity conditioning with fludarabine and treosulfan prior to allogeneic stem cell transplantation in multiple myeloma.

In recent years, reduced-intensity conditioning (RIC) regimens before allogeneic stem cell transplantation (SCT) are increasingly used in patients not eligible for conventional conditioning. We did a retrospective, multicenter analysis to assess the feasibility of conditioning with fludarabine and treosulfan before allogeneic SCT in multiple myeloma patients. Thirty-four patients with a median age of 51.5 years were included in the analysis. All patients underwent myeloablation after conditioning followed by stable engraftment, and 29 of 31 evaluable patients (94%) showed early complete hematopoietic chimerism. Non-hematological toxicities were limited and encompassed mainly fever in neutropenia and infections. Grade II-IV acute and chronic graft-versus-host disease was observed in 33 and 39%, respectively. With a median follow-up of 708 days (range 60-1729 days), the median progression-free survival was 180 days. The treatment-related mortality was 10% on day 100 and 25% after 1 year. The median overall survival has not yet been reached. Our data indicate that conditioning with fludarabine and treosulfan before allogeneic SCT is feasible in intensively pretreated multiple myeloma patients and leads to stable engraftment and complete hematopoietic chimerism. Randomized trials are warranted to determine if this approach might be incorporated in an algorithm of multiple myeloma treatment.

Autologous blood stem-cell transplantation in patients with advanced Hodgkin's disease and prior radiation to the pelvic site.

Patients with relapsed Hodgkin's disease who respond to salvage therapy are successfully treated with cyclophosphamide, carmustine (BCNU), and etoposide (VP-16) (CBV) followed by autologus bone marrow transplantation (ABMT). Because of heavy pretreatment including radiation to the pelvic site, marrow harvest was not feasible in those patients. We therefore used blood-derived hemopoietic precursor cells as an alternative stem-cell source to rescue them after superdose chemotherapy. Hemopoietic precursor cells were mobilized into the peripheral blood either by chemotherapeutic induction of transient myelosuppression followed by an overshooting of blood stem-cell concentration, or by continuous intravenous (IV) granulocyte-macrophage colony-stimulating factor (GM-CSF) administration. The median time to reach 1,000 WBC per microliter, 500 polymorphonuclear cells (PMN) per microliter, or 20,000 platelets per microliter was 10, 20.5, and 38 days, respectively, for 50% of all patients. The platelet counts of two patients never dropped below 20,000/microL following autologous blood stem-cell transplantation (ABSCT), whereas two other patients had to be supported with platelets for 75 and 86 days posttransplant until a stable peripheral platelet count of 20,000/microL was attained. Among the 11 assessable patients, seven are in unmaintained complete remission (CR) at a median follow-up of 318 days. This is a first report on a series of ABSCTs in patients with advanced Hodgkin's disease proving that, despite prior damage to the marrow site, the circulating stem-cell pool is still a sufficient source of hemopoietic precursor cells for stem-cell rescue.

Is there a place for 2-CDA in the treatment of B-CLL?

There is no doubt about 2-CDA being a very potent lymphotoxic agent that displays high efficacy in the treatment of CLL. It interferes with the intranuclear machinery of DNA regulation, and causes death to proliferative active, as well as resting lymphocytes. Interruption of crucial pathways that are evident for cell survival translates into high clinical response rates in CLL. CR and PR rates comparable to those reported on fludarabine are achieved in relapsed or refractory CLL. Even though trials on previously untreated CLL are still ongoing, a consistent trend towards durable, high CR rates becomes apparent. The toxicity is comparable to that of fludarabine and consists of infections, as well as thrombocytopenia. Clinical as well as in vitro studies suggest a crossresistance between the two purine analogues, indicating that sequential treatment is not useful. Given these data, although preliminary in case of de novo CLL, 2-CDA has to be recognized as one of the most effective cytostatic drugs currently available for CLL treatment. Large prospective trials (in comparison with fludarabine) will assess the role of 2-CDA as standard treatment. Such trials should also have the aim to substantiate the potential of 2-CDA as induction treatment followed by high-dose consolidation.

Plasma levels of soluble CD8 antigen and interleukin-2 receptor antigen in patients with hairy cell leukemia, relationship with splenectomy and with clinical response to therapy.

A soluble form of CD8 antigen (sCD8) has been shown to be released by activated CD8 + lymphocytes. Measurements of sCD8 may serve as an index of suppressor/cytotoxic cell activity. To assess the clinical significance of this observation in response to malignancy, we have investigated the sCD8 concentrations in 38 patients with hairy cell leukemia (HCL) not yet treated with any systemic therapy. The median plasma sCD8 level of the 20 nonsplenectomized patients was 1,025 U/ml and was significantly higher than that in the 18 patients who had previous splenectomy (median = 200 U/ml, p less than 0.0001), or in 14 normal controls (median = 350 U/ml, p less than 0.0001). Compared to controls, splenectomized patients had also significantly lower levels of sCD8 (p less than 0.01). The median concentration of soluble interleukin-2 receptor (sIL2R) in nonsplenectomized patients was 14,500 U/ml and was in the same range as in splenectomized patients (15,000 U/ml). There was no overlap in sIL2-R levels between controls (median = 300 U/ml) and patients. Investigation of serial plasma samples in 7 patients who received deoxycoformycin (DCF) and 11 patients treated with interferon alpha (IFN-alpha) showed a normalization of sCD8 levels and a decrease of sIL2R concentrations in those patients who showed hematological improvement. Normalization of sIL2R was, however, only observed in patients with complete remission. Our observation indicates that splenectomy might cause a reduction of the activation of suppressor/cytotoxic cells in patients with HCL. Treatment with either DCF or IFN-alpha also modulates the sCD8 levels to normal range. Measurements of sCD8 and sIL2-R might give more insight into the pathogenesis of HCL and serve as parameters for monitoring different phases of the disease and response to therapy.

Nonmyeloablative stem cell transplantation in adults with high-risk ALL may be effective in early but not in advanced disease.

The feasibility of nonmyeloablative stem cell transplantation (NST) was evaluated in 22 adults with high-risk ALL. 16/22 patients had advanced disease and 11/22 had Ph+ ALL. Eleven patients received NST as first stem cell transplantation (SCT). Eleven patients had relapses after allogeneic or autologous SCT and underwent a salvage NST. 18/22 patients (82%) engrafted after NST. 13/16 patients (81%) with active disease reached complete remission (CR). 11 of 13 patients developed GVHD. After first NST 10/11 patients (91%) engrafted. Six of seven patients with active disease reached CR. Three of five relapsing patients reached subsequent CR after donor lymphocyte infusions, termination of immunosuppression or imatinib. Three of 11 patients (27%) are alive in CR 5 to 30 months after NST. Eight of 11 patients have died, 3/8 from leukemia and 5/8 from transplant-related causes. After salvage NST, 8/11 patients (73%) engrafted. Seven of nine patients with active disease reached CR. Only one of 11 patients transplanted, who was in CR before undergoing salvage NST is alive 19 months after NST. Five of 11 have died from leukemia, one of 11 after graft failure and four of 11 from transplant-related causes. Four of 22 patients (18%) are alive in CR 5, 14, 19 and 30 months after NST. NST is feasible in adults with high risk ALL. However, transplant-related mortality remains high and only patients transplanted in CR seem to have long-term disease-free survival.

Efficacy and safety of simultaneous immunomagnetic CD34+ cell selection and breast cancer cell purging in peripheral blood progenitor cell samples used for hematopoietic rescue after high-dose therapy.

We have established a new simultaneous positive/negative selection procedure using the Baxter Isolex 300i system. We tested its tumor cell (TC) purging efficacy by tumor contamination tests ex vivo and its safety in a group of 17 breast cancer (BC) patients by measuring hematopoietic recovery after high-dose (HD) therapy and autologous stem cell rescue with the selected cells. Tumor contamination tests resulted in a TC depletion of 4.1-6.0 log steps. The CD34+ cell yield in this experimental setting was 38.9-91.5%, and the CD34+ cell purity was 86.0-96.0%. In a group of 17 BC patients (5 high-risk adjuvant, > or = 10 lymph nodes positive, and 12 metastatic), we processed leukapheresis products (LPs) by simultaneous positive/negative selection. In these clinical samples, the mean CD34+ cell yield was 56.2% (range, 14.0-80.1%), and the CD34+ cell purity was 94.5% (range, 69.0-99.8%). Additionally, we screened samples of the patients' LPs before and after the purging procedure for contaminating TC by immunocytochemistry. In 15 of 17 tested cases, TCs were detectable prior to the purging procedure. After the procedure, we could not detect residual TCs in 16 of 17 cases. In one case, we found a highly reduced number of TCs. Furthermore, we evaluated the times for hematopoietic reconstitution in a group of five BC patients in the high-risk adjuvant situation who underwent HD chemotherapy and hematopoietic rescue with positive/negative selected stem cells and compared it with our own data from 10 BC patients who, after identical HD therapy, received only positively selected CD34+ cells and 14 patients who, after identical HD therapy, received autografts purged by incubation with toxic ether lipids (ET-18-OCH3). In all groups, a leukocyte count of >2000 cells/microl was reached at day +10. A platelet count of > 50,000 cells/microl was reached at day +12 in the ET-18-OCH3 group and at day +14 in the other two groups. Furthermore, 12 patients with metastatic disease rescued with positive/negative selected stem cells after HD therapy also showed fast and comparable hematopoietic recovery. The new simultaneous immunomagnetic positive/negative selection using a closed system is effective and safe. Processing LPs leads to a similar CD34+ cell yield, a higher TC depletion compared to standard CD34+ cell selection, and no delay in hematopoietic recovery.

Successful autologous transplantation of blood stem cells mobilized with recombinant human granulocyte-macrophage colony-stimulating factor.

We investigated the effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) on the pool of circulating hemopoietic progenitor cells in 11 patients with hematological malignancies of nonmyeloid origin and 1 patient with sarcoma. These patients were eligible for autologous blood stem cell transplantation rather than autologous bone marrow transplantation because sufficient marrow aspirates could not be performed due to damage at the usual sites of bone marrow harvest by previous chemo- and/or radiotherapy. Recombinant human GM-CSF was given as continuous i.v. infusion via central venous line for a median time of 11.5 days (range 5-22 days), during which a median number of six aphereses were performed. In comparison to the pretreatment level the median increase in the number of granulocyte-macrophage colony-forming units (CFU-GM)/ml of peripheral blood was 8.5-fold. In all 12 patients a median decrease of the platelet count of 21% (range 7%-67%) was observed during rhuGM-CSF treatment prior to the start of the apheresis procedures. Six patients were treated with a myeloablative conditioning therapy consisting of total body irradiation and/or high-dose polychemotherapy followed by autografting with blood stem cells. Five of them achieved a sustained engraftment. Recombinant human GM-CSF proved to be highly efficient in increasing the number of circulating progenitor cells in these patients with severely compromised hemopoiesis. Blood stem cells harvested under a rhuGM-CSF treatment are capable of restoring hemopoiesis in man after a myeloablative pretransplant therapy.

A phase I trial of interferon alpha-2b with folinic acid and 5-fluorouracil administered by 4-hour infusion in metastatic colorectal carcinoma.

Preclinical data suggest that both folinic acid and interferon may enhance the efficacy of 5-fluorouracil (5-FU) in colorectal carcinoma. We therefore initiated a phase I trial evaluating the doses, safety, and pharmacokinetics of the combination of recombinant interferon (IFN) alpha-2b with folinic acid (FA) and 5-FU. Seventeen patients with colorectal cancer who failed local chemotherapy received 5-FU as a 4-hour infusion, preceded by a bolus of FA and IFN. The 5-FU dose was escalated over the range of 400 to 650 mg/m2/d for a period of 7 days. Folinic acid was administered as a bolus in a fixed dose of 200 mg/m2/d and IFN as 5 million U/d subcutaneously on days 1 to 7. A total of 89 courses of therapy were completed for the 17 patients, of which there were 10 paired courses with a combination of 5-FU and IFN or 5-FU alone, being performed to analyze the pharmacokinetics and modulation of 5-FU by IFN. The maximum tolerated dose of 5-FU using this combination and a 4-hour schedule was 600 mg/m2/d for 7 days. The dose-limiting toxicity of this regimen was diarrhea. Mucositis and myelosuppression was not a marked problem at dose levels of 400 and 500 mg/m2/d for 7 days. However, at a dose level of 600 to 650 mg/m2/d for 7 days, grade 3 and 4 (WHO) leukopenia occurred in 50% and mucositis occurred in 33%. At a given dose of 5 million U, IFN did not significantly influence 5-FU serum levels. Mean steady-state serum levels of 5-FU at 500 mg/m2 given as a 4-hour infusion were 16.55 +/- 9.34 mumol/L and 18.23 +/- 12.77 mumol/L with and without IFN, respectively. Mean area under the curve (mumol/L x min) was 4,008 +/- 2,133 and 5,114 +/- 2,567 with and without interferon, respectively. Objective responses were seen in one of 17 of these heavily pretreated patients and stable disease was seen in seven of 17 patients. The recommended dose of 5-FU for use of phase II studies is 500 mg/m2/d for 7 days. We conclude that the toxicity of 5-FU plus FA with and without IFN alpha-2b can be reduced by using a 4-hour infusion instead of a bolus.

Detection of circulating monoclonal lymphocytes in multiple myeloma patients by analysis of gene rearrangements: correlation with progressive disease.

Multiple myeloma is characterized by the proliferation of a monoclonal plasma cell population that essentially spreads in the bone marrow. However, immunological data and studies on clonal gene rearrangements have provided evidence for circulating malignant B-lymphocytes. Herein, we report on the detection and clinical significance of monoclonal lymphocytes in the peripheral blood of multiple myeloma patients. Applying the analysis of immunoglobulin gene rearrangements, clonal circulating lymphocytes were found in 8 out of 37 patients. In three of these eight patients, DNA from the bone marrow could be examined as well, and the same gene rearrangement as in peripheral blood was detected. This observation indicates that the monoclonal lymphocytes in the peripheral blood are part of the malignant plasma cell clone in the bone marrow. There was a strong correlation between progressive stage III disease and the detection of circulating tumour cells, whereas neither in stable stage III nor stage I disease could clonal gene rearrangements be detected. Our findings indicate the high incidence of monoclonal lymphocytes in the peripheral blood from patients with progressive multiple myeloma. This may be of importance with respect to cell harvesting strategies for autologous peripheral stem cell transplantation in multiple myeloma.

Combination of low-dose cytarabine and 13-cis retinoic acid in the treatment of myelodysplastic syndromes.

Responses have been reported in patients with myelodysplastic syndromes (MDS) after low-dose cytarabine (Ara-C) or 13-cis-retinoic acid (13-CRA) therapy. Recently, combination of these two substances in vitro was shown to produce a synergistic effect on differentiation of leukemic cells. We conducted a phase II trial with low-dose Ara-C (5 mg/m2 per 12 h s.c.) and 13-CRA (60 mg/m2 per day orally) in 14 patients with MDS, six of whom had refractory anemia with excess of blasts (RAEB), seven had RAEB in transformation (RAEBt) and one chronic myelomonocytic leukemia (CMML). The drugs were administered from day 1 to 14 and the treatment courses repeated every 4 to 8 weeks. One partial response and one minor response could be achieved. Major toxicity included dry skin, mucositis and cheilitis in 11 of the 14 patients. The response rate is no better than the results reported in the literature with either drugs alone. As yet there is no satisfactory treatment for MDS.

Enzyme activities of leukemic cells and biochemical changes induced by deoxycoformycin in vitro--lack of correlation with clinical response.

Deoxycoformycin (DCF) is a specific inhibitor of adenosine deaminase (ADA) and has been shown to be active in lymphoid neoplasms. Cytotoxicity is thought to be mediated by the accumulation of deoxyadenosine (AdR) and deoxyadenosine triphosphate (dATP) which inhibits ribonucleotide reductase and DNA synthesis in rapidly proliferating cells. Others suggested mechanisms leading to cell death particularly in non-dividing cells include depletion of ATP and NAD pools, inhibition of S-adenosylhomocysteine (SAH) hydrolase and induction of DNA strand breaks. In patients with high leukemic counts who were subsequently treated with DCF, we have studied (a) the levels of ADA, ecto-5'-nucleotidase (5NT), deoxyadenosine kinase (AdR-kinase) and SAH-hydrolase in the leukemic cells; [b) the in-vitro effects of DCF on dATP, ATP, NAD, SAH-hydrolase levels and on DNA strand breaks; and (c) the correlation between these parameters with clinical response to DCF. No significant difference in ADA, 5NT, AdR-kinase and SAH-hydrolase activities could be found between responders and non-responders. Incubation of the leukemic cells in vitro with DCF caused an inhibition of ADA, an accumulation of dATP, a moderate reduction in ATP and NAD levels, a suppression of SAH-hydrolase activity and an increase in DNA strand breaks in practically all the leukemic samples, irrespective of clinical response. Our results show that neither measurement of these enzymes nor studies of these biochemical sequelae of ADA inhibition in vitro predicts clinical responsiveness to DCF therapy.

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 x 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 x 10(6)/kg CD34+ cells were transplanted. The number of CD3+ cells in the allografts was 5.7 x 10(4)/kg (median) after Isolex 300i, and 0.2 x 10(4)/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 x 10(6)/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 x 10(6)/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.

Quantitative real-time RT-PCR detects elevated Wilms tumor gene (WT1) expression in autologous blood stem cell preparations (PBSCs) from acute myeloid leukemia (AML) patients indicating contamination with leukemic blasts.

High-dose chemotherapy with subsequent autologous stem cell transplantation is believed to be of therapeutic benefit in patients with acute myeloid leukemia (AML), especially when no allogeneic bone marrow donor is available. One of the main risks is contamination of the stem cell preparations with leukemic blasts, which may account for a higher relapse rate compared to allogeneic bone marrow transplantation. Since overexpression of WT1 is common in leukemic blasts, we investigated, whether PBSCs from AML patients express WT1 at a higher level as compared to patients with solid cancers. PBSCs of seven patients with AML and of five patients with solid cancers were investigated for WT1 expression. Total WT1 copy count was determined in a standardized quantitative real time RT-PCR. WT1 expression was found in all AML PBSCs with an average copy number of 49.99 +/- 61.09. In solid cancers WT1 expression was statistically significantly lower with a copy number of 3.51 +/- 1.92. In AML patients with sustained complete remission we found a nearly significantly lower WT1 expression than in patients who relapsed within the first year after stem cell transplantation. Our data show a higher WT1 expression in PBSCs of AML patients compared to patients with solid cancers. This finding might indicate a contamination with leukemic blasts. Quantification of WT1 in PBSCs might therefore be useful to estimate the risk of relapse after autologous stem cell transplantation in AML patients.

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.

Chemopurging of peripheral blood-derived progenitor cells by alkyl-lysophospholipid and its effect on haematopoietic rescue after high-dose therapy.

One reason for relapse after high-dose tumor therapy with subsequent autologous stem cell transplantation is tumor cell contamination of the graft. Removal of tumor cells from bone marrow grafts by chemopurging with the ether lipid edelfosine has been established as an effective and simple method. When compared with bone marrow derived grafts, progenitor cells from peripheral blood have considerably reduced the haematological recovery times. However, this advantage is put at risk by the nonspecific haematotoxic activity of the purging agent. We therefore compared the in vitro recovery of peripheral blood derived progenitor cells (PBPC) from either non-purged (n = 41) or purged (75 micrograms/ml of ether lipid for 4 h at 37 degrees C, n = 48) leukapheresis products. The recovery of CFU-GM after cryopreservation was 63 +/- 4% without and 48 +/- 3% with purging (P = 0.007). After high-dose therapy, patients (n = 37) received similar amounts of either non-purged (n = 17) or purged (n = 20) autologous PBPC. The median haematological recovery times (non-purged vs purged) to > 500 WBC/microlitres were 9.0 vs 8.5 days after transplantation, to > 2000 PMN/microlitres 10.5 vs 10.0 days, and to > 50,000 PLT/microlitres 15.5 vs 14.0 days. All differences were statistically not significant. We conclude that ether lipid purging of PBPC leads to a significant, however tolerable loss of progenitor cells in vitro, and that haematological recovery times after high-dose therapy are identically short, provided similar amounts of PBPC are reinfused.

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, II-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.