Prior to purging, cryopreservation, or ABO-incompatible bone marrow (BM) transplantation, we have concentrated 23 BM harvests using a modification of the "grancollect-protocol" and the recently available bone marrow stem cell (BMSC) protocol of the Fresenius AS 104 cell separator with the P1-Y set. Within 40-70 minutes, the initial marrow volume of 922 ml (+/- 408 ml) was processed two to three times. A mean of 59% (+/- 20%) of the initial mononuclear cells was recovered in a mean volume of 119 ml (+/- 31 ml). The recovery of clonogenic cells, measured by CFU-GM assays, was 98% (+/- 80%). Red blood cells in the BM concentrates were reduced to 8% (+/- 4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation, and transplantation. All transplanted patients showed fast and sustained engraftments after autologous or allogeneic BM transplantation.

Chemie Ingenieur TechnikVolume 78, Issue 9 p. 1382-1383 VortragFree Access Komponenten aus Mikroalgen mit Aktivitäten gegen humanpathogene Viren T. König, T. König tanja.koenig@bvt.cbi.uni-erlangen.de Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorS. Thulke, S. Thulke Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorI. Naumann, I. Naumann Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorW. Siegert Prof., W. Siegert Prof. Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorR. Buchholz Prof., R. Buchholz Prof. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorC. Walter Dr., C. Walter Dr. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this author T. König, T. König tanja.koenig@bvt.cbi.uni-erlangen.de Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorS. Thulke, S. Thulke Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorI. Naumann, I. Naumann Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorW. Siegert Prof., W. Siegert Prof. Medizinische Klinik II m. S. Onkologie und Haematologie, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 BerlinSearch for more papers by this authorR. Buchholz Prof., R. Buchholz Prof. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this authorC. Walter Dr., C. Walter Dr. Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schloßplatz 4, D-91054 ErlangenSearch for more papers by this author First published: 06 September 2006 https://doi.org/10.1002/cite.200650194Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1 C. Walter, T. Steinau, N. Gerbsch, R. Buchholz, Biomol. Eng. 2003, 20 (4–6), 261. 2 S. Rechter et al., Jahrestagung der Gesellschaft für Virologie, München 2006. Citing Literature Volume78, Issue9Special Issue: GVC/DECHEMA-Jahrestagungen 2006 mit 24. DECHEMA-Jahrestagung der BiotechnologenSeptember, 2006Pages 1382-1383 ReferencesRelatedInformation

The aim of this study was assess the activity of thalidomide in patients with progressive relapsed or platinum-refractory germ-cell tumours (GCT). Between April 2002 and January 2003, 15 patients with inoperable progressive GCT were treated with escalated daily doses of 200-600 mg thalidomide. All patients had failed first-line and salvage chemotherapy with a median of 6 (range 4-12) cisplatin-based treatment cycles, 13/15 (87%) patients had received high-dose chemotherapy (HDCT) and 8/15 (53%) patients were considered platinum-refractory or absolute refractory; 8/15 (53%) patients had previously received other palliative chemotherapy regimens. No patient achieved a complete remission (CR) or partial remission (PR). However, 5/15 (33%) patients achieved serological PR and 1 additional patient had stable disease for 3 months. The median duration of remissions was 3 months (range 2-12 months) including 2 patients with a progression-free survival of 9 and 12 months. Responses occurred mainly in patients with a low tumour burden, slow disease progression and alpha-foetoprotein (AFP) elevations. Responses to thalidomide were independent from platinum-sensitivity. Toxicity was mild, with lethargy and constipation in the majority of patients. Skin rash grade II developed in 2 patients and peripheral neurotoxicity grade II/III developed in 4 patients. One responding patient died suddenly from an unknown cause. It is concluded that thalidomide shows single-agent activity in patients with heavily pre-treated GCT, AFP elevations and slowly progressive disease.

Background. Recombinant methionyl granulocyte colony-stimulating factor (methuG-CSF; Filgrastim) is commonly used to reduce the duration of neutropenia that follows allo BMT performed after high-dose chemo-/radiotherapy (HDT) in pts with HM. Recently, a retrospective chart review suggested that leukemic pts treated with G-CSF after allo BMT had faster neutrophil recovery, but slower platelet engraftment, increased risk of graft vs host disease (GvHD), and reduced survival relative to pts who did not receive G-CSF (Ringdén et al, 2004). The present double-blind, randomized, placebo-controlled, multicenter phase 3 study was designed to evaluate the effects of Filgrastim treatment on neutrophil recovery. Pts were to be followed for 2 years.

BACKGROUND: Isohemagglutinins directed against the donor blood group frequently delay erythroid engraftment after major ABO-mismatched allogeneic hematopoietic progenitor cell transplantation (HPCT). Graft-versus-host reactions are capable of accelerating the clearance of isohemagglutinins. Whether immunogenicity of the A- and B-antigen is important in this process is unknown.PATIENTS AND METHODS: Data of 807 patients from three centers were screened for patients with major or bidirectionally ABO-mismatched donors. Clinical data and red blood cell (RBC) transfusion support were analyzed retrospectively.RESULTS: A total of 158 patients with major or bidirectionally mismatched donors were identified. After major mismatched HPCT, patients with anti-A directed against the donor blood group required RBC transfusion support for a median of 109 days (range, 0-324 days) compared to 21 days (range, 2-98 days) for patients with anti-B directed against donor blood group (log-rank test, p = 0.0001). Other risk factors associated with prolonged RBC transfusion support in univariate analysis were age (p = 0.024), cytomegalovirus infection (p = 0.016), hemolytic anemia (p = 0.027), and chronic bleeding disorders (p = 0.038). The independent influence of donor blood group and recipient age were confirmed in a multivariate analysis.CONCLUSION: These results indicate that the immunogenicity of the ABO antigen plays an important role for the kinetics of erythroid engraftment after ABO-mismatched HPCT.

The clinical significance of human herpesvirus (HHV-6) infections after allogeneic stem cell transplantation (SCT) remains controversial. We analysed cryoconserved plasma samples from 82 patients after allogeneic SCT by quantitative polymerase chain reaction for HHV-6 variants A and B. Platelet engraftment was delayed in patients with HHV-6B infections but not with HHV-6A infections detected before day +28. In multivariate analysis early HHV-6B infections and the type of conditioning were associated with platelet engraftment. In conclusion, the two variants of HHV-6 should be studied separately; early infections with HHV-6B may contribute to delayed platelet engraftment after allogeneic SCT.

BACKGROUNDIncomplete remission or relapse from first-line chemotherapy has poor prognosis in male germ cell tumour patients. This phase III randomised trial compares conventional salvage to high-dose-intensification chemotherapy.PATIENTS AND METHODSBetween February 1994 and September 2001, 280 patients from 43 institutions in 11 countries, were randomly assigned to receive either four cycles of cisplatin, ifosfamide and etoposide (or vinblastine) (arm A), or three such cycles followed by high-dose carboplatin, etoposide and cyclophosphamide (CarboPEC) with haematopoietic stem cell support (arm B).RESULTSSimilar complete and partial response rates were observed in both treatment arms (56%; 95% CI 50% to 62%). There were 3% and 7% toxic deaths in arms A and B, respectively. No significant improvements with CarboPEC were observed in either 3-year event-free survival (35% versus 42%, P=0.16) or overall survival (53%; 95% CI 46% to 59%). Complete responders with CarboPEC had a significant improvement in disease-free survival (55% versus 75% at 3 years, P <0.04).CONCLUSIONSThe single cycle of high-dose salvage chemotherapy after three cycles of standard dose chemotherapy had no effect on treatment outcomes. These results suggest that data from uncontrolled studies should not be used to justify routine use of a toxic and expensive treatment without confirmation in a randomised trial.

PURPOSE In the era of molecular therapy of chronic myelogenous leukemia (CML) applying BCR-ABL tyrosine kinase inhibitors, the usefulness of molecular end points, in particular, quantitative polymerase chain reaction (PCR) for BCR-ABL in monitoring responses has been broadly accepted. Therefore, we have designed a prospective phase II trial in CML, which, for the first time, evaluated the feasibility and safety of molecular end points as surrogate markers to guide through a stratified treatment algorithm within a multicenter trial. PATIENTS AND METHODS As a clinical model, we adopted minimal residual disease (MRD) found in relapse after allogeneic stem cell transplantation (SCT) in CML. Forty-four patients were enrolled and received the BCR-ABL tyrosine kinase inhibitor imatinib (IM) at a starting dose of 400 mg/d. The quality of molecular responses achieved then decided on discontinuation of IM or dose escalation up to 800 mg/d, and finally, on application of donor lymphocyte infusions. Results Seventy percent of patients achieved a complete molecular response (CMR), defined as nested PCR-negativity for BCR-ABL in three consecutive samples. Interestingly, in four out of 10 patients who discontinued IM, CMR was durable even after cessation of IM with a median follow-up of 494 days. This suggests the possibility of long-term tumor control in a subset of patients. CONCLUSION The treatment strategy showed that IM treatment was well-tolerated and highly efficacious in MRD after allogeneic SCT. Moreover, this study demonstrated that evaluation of a molecular end point within a multicenter trial can be a safe and effective tool for clinical decision making.

BACKGROUND Results of second-line chemotherapy in patients with extragonadal non-seminomatous germ cell tumor (NSGCT) appear inferior to results in testicular NSGCT. Patients with retroperitoneal NSGCT achieve a comparable long-term survival rate of 30%, but the salvage rates of patients with mediastinal primary are less than 10%. We conducted a retrospective analysis on patients with mediastinal and retroperitoneal NSGCT treated with second-line high-dose chemotherapy (HDCT) registered with the European Group for Blood and Marrow Transplantation (EBMT). PATIENTS AND METHODS Between 1987 and 1999, 59 registered patients with retroperitoneal (n=37) and mediastinal (n=22) primary NSGCT, median age 28 years (range 18-60), were treated with second-line HDCT. All had received cisplatin-containing chemotherapy as first-line treatment. RESULTS Toxic death occurred in three cases (5%). With a median follow-up of 58 months (range 14-114), 18/59 patients (30%) continue to be disease-free. Of three patients who had a disease recurrence after HDCT, one patient achieved a disease-free status with further chemotherapy and surgery. In total, 19 patients (32%) are currently disease-free. Sixteen of 37 patients (43%) with retroperitoneal NSGCT, and three of 22 patients (14%) with mediastinal NSGCT are currently alive and disease-free. CONCLUSIONS Second-line HDCT might represent a possible option for patients with retroperitoneal primary NSGCT. New salvage strategies are needed for patients with mediastinal NSGCT.

PURPOSETo assess the role of residual tumor resection performed after high-dose chemotherapy (HDCT) in patients with relapsed or refractory germ cell tumors (GCT).PATIENTS AND METHODSBetween July 1987 and October 1999, postchemotherapy resections of residual tumors were performed in 57 patients who had been treated with HDCT for relapsed or refractory GCT and who had achieved a partial remission to this treatment.RESULTSComplete resections of residual masses were achieved in 52 (91%) of 57 patients who were rendered disease free; in five (9%) of 57 patients, the resections were incomplete. Resection of a single site was performed in 39 (68%) of 57 patients, and the remaining 18 (32%) of 57 patients required interventions at two or more residual tumor sites. Necrosis was found in 22 (38%) of 57 patients, mature teratoma with or without necrosis was found in nine (16%) of 57 patients, and viable cancer with or without additional necrosis or mature teratoma was found in 26 (46%) of 57 patients. Viable cancer consisted either of residual germ cell or undifferentiated cancer in 22 (85%) of 26 patients, with additional non-GCT histologies in the remaining four patients. Patients with viable cancer had a significantly inferior outcome after surgery compared with patients with necrosis and/or mature teratoma even if all cancer was completely resected. Pulmonary lesions with a diameter of more than 2 cm were the only predictive variable for viable cancer in univariate analysis.CONCLUSIONResections of all residual tumors should be attempted in patients with relapsed or refractory GCT and partial remissions after HDCT.

Recently we demonstrated that RIC with busulfan, fludarabine and ATG followed by allogeneic hematopoietic stem cell transplantation (HSCT) induced molecular remissions in patients (pts) with advanced CLL. However, this approach was hampered by severe GVHD. In an attempt to lower the rate of severe GVHD we replaced ATG by campath in a new study protocol.

Objective: quantitative evaluation of viruria (DNA-uria) and hematuria in patients with SCT and analyze both relationship clinically.

To elucidate the role of human herpesvirus 6 (HHV-6) in hematopoiesis, the influence of HHV-6A and HHV-6B on the in vitro expansion and differentiation of cord blood (CB) progenitor cells was investigated in liquid culture. Nonfractionated CB mononuclear cells (CB-MNC) or MACS-enriched CD34+ CB cells were seeded in liquid culture under conditions allowing maximal expansion of nucleated cells. Cells were either incubated with HHV-6A- or HHV-6B-containing cell culture supernatants or a virus-free control. After 7, 14, and 21 days, cells were analyzed for growth by cell count, for differentiation by flow cytometry, and for HHV-6 infection by antigen detection or PCR. Expansion of CB-MNC was significantly inhibited by HHV-6A and HHV-6B for a period of 3 weeks, including reduced proportions of CD34+ and CD33+ cells in HHV-6-treated cultures on day 7. In contrast, when starting with enriched CD34+ cells, the expansion was only affected by HHV-6A. Inhibition of CD34 and CD33 cell development was less pronounced in these cultures compared to CB-MNC cultures. However, HHV-6 antigen and DNA was detectable in these cultures. In conclusion, although HHV-6A inhibited expansion of CD34 progenitor cells, HHV-6B inhibited growth of immature CB cells only in interaction with nonfractionated CB-MNC.

Purpose: In a phase II clinical trial we have previously reported on the safety and efficacy of imatinib mesylate (IM) to induce hematologic, cytogenetic and molecular remissions in case of relapse post allogeneic stem cell transplantation (SCT) in patients with chronic myelogenous leukaemia (CML). Here we report on an extended follow-up phase, which was performed to monitor stability of responses and further disease course in patients enrolled. Patients and Methods: Within the trial, patients, transplanted in chronic phase (CP) CML with molecular or cytogenetic relapse (n=37), received IM at a starting dose of 400mg. Close monitoring was performed, which, besides evaluation of side effects, included hematology, chimerism, bone marrow analysis and quantitative/qualitative PCR for Bcr-Abl. After completion of the study phase, pts were treated by the discretion of their physician, which could include continuation or cessation of IM or application of DLI. Results: During the entire observation period the rate of reported side effects was low, only one mild reactivation of Graft versus Host Disease (GvHD) occurred upon IM treatment. Response rates reflected high efficacy of IM in this patient population: in 16/22 (72.7%) evaluable patients a complete chimerism in peripheral blood was demonstrated. Complete cytogenetic response (CCR) was seen in 11/13 (84.6%) of patients with cytogenetic relapse and follow-up samples available. Importantly, 25/37 patients (67%) achieved a complete molecular response (CMR) (defined as nested PCR-negativity for 3 consecutive samples) within the primary study phase or during follow-up. Cessation of IM in 10 patients with CMR during follow-up resulted in molecular relapse of 6 patients (60%). However, importantly in 4/10 (40%) patients, CMR were durable even after cessation of the study drug with a median follow-up of 381 days after discontinuation of IM. In 7 patients, donor lymphocyte infusions (DLI) were given without major toxicities. This established CMR in 4 of these patients. One episode of mild GvHD reactivation did occur in the context of DLI. At the median follow-up of 1.7 years, OS is 100% in the study cohort; one patient has died due to progressive disease 2 years after inclusion into the study. Conclusion: In CP-CML patients with relapse post allogeneic SCT IM treatment established CMR in a majority of patients. Importantly, CMR were noted even after cessation of the study drug, suggesting the possibility of definite tumor control in selected patients. Therefore, weighing the risk of aggravation of GvHD and/or induction of bone marrow aplasia using DLI against the favourable toxicity profile of IM, in case of MRD post allogeneic SCT relapse and primary IM-treatment seems to be justified. In particular in patients with poor performance status or active GvHD early therapy instead of postponed treatment may lead to superior results. Further clinical trials are warranted to optimize sequential or concurrent use of IM and DLI in this patient population.

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and β-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that “Classical” reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.