Allogeneic stem cell transplantation for patients with advanced rhabdomyosarcoma: a retrospective assessment.

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-SCT) may provide donor cytotoxic T cell-/NK cell-mediated disease control in patients with rhabdomyosarcoma (RMS). However, little is known about the prevalence of graft-vs-RMS effects and only a few case experiences have been reported. METHODS: We evaluated allo-SCT outcomes of 30 European Group for Blood and Marrow Transplantation (EBMT)-registered patients with advanced RMS regarding toxicity, progression-free survival (PFS) and overall survival (OS) after allo-SCT. Twenty patients were conditioned with reduced intensity and ten with high-dose chemotherapy. Twenty-three patients were transplanted with HLA-matched and seven with HLA-mismatched grafts. Three patients additionally received donor lymphocyte infusions (DLIs). Median follow-up was 9 months. RESULTS: Three-year OS was 20% (s.e.±8%) with a median survival time of 12 months. Cumulative risk of progression was 67% (s.e.±10%) and 11% (s.e.±6%) for death of complications. Thirteen patients developed acute graft-vs-host disease (GvHD) and five developed chronic GvHD. Eighteen patients died of disease and four of complications. Eight patients survived in complete remission (CR) (median: 44 months). No patients with residual disease before allo-SCT were converted to CR. CONCLUSION: The use of allo-SCT in patients with advanced RMS is currently experimental. In a subset of patients, it may constitute a valuable approach for consolidating CR, but this needs to be validated in prospective trials.

Long-term follow-up of high-dose chemotherapy with autologous stem-cell transplantation and response-adapted whole-brain radiotherapy for newly diagnosed primary CNS lymphoma: results of the multicenter Ostdeutsche Studiengruppe Hamatologie und Onkologie OSHO-53 phase II study.

BACKGROUND: We previously reported the results of a phase II study for patients with newly diagnosed primary central nervous system lymphoma treated with autologous peripheral blood stem-cell transplantation (aPBSCT) and response-adapted whole-brain radiotherapy (WBRT). Now, we update the initial results. PATIENTS AND METHODS: From 1999 to 2004, 23 patients received high-dose methotrexate. In case of at least partial remission, high-dose busulfan/thiotepa (HD-BuTT) followed by aPBSCT was carried out. Patients refractory to induction or without complete remission after HD-BuTT received WBRT. Eight patients still alive in 2011 were contacted and Mini-Mental State Examination (MMSE) and the European Organisation for Research and Treatment of Cancer quality-of-life questionnaire (QLQ)-C30 were carried out. RESULTS: Of eight patients still alive, median follow-up is 116.9 months. Only one of nine irradiated patients is still alive with a severe neurologic deficit. In seven of eight patients treated with HD-BuTT, health condition and quality of life are excellent. MMSE and QLQ-C30 showed remarkably good results in patients who did not receive WBRT. All of them have a Karnofsky score of 90%-100%. CONCLUSIONS: Follow-up shows an overall survival of 35%. In six of seven patients where WBRT could be avoided, no long-term neurotoxicity has been observed and all patients have an excellent quality of life.

Primary central nervous system lymphoma treated with high-dose methotrexate, high-dose busulfan/thiotepa, autologous stem-cell transplantation and response-adapted whole-brain radiotherapy: results of the multicenter Ostdeutsche Studiengruppe Hamato-Onkologie OSHO-53 phase II study.

BACKGROUND: We investigated the efficacy and safety of tandem high-dose methotrexate (HD-MTX) induction followed by high-dose busulfan/thiotepa (HD-BuTT) with autologous peripheral blood stem-cell transplantation (aPBSCT) and response-adapted whole-brain radiation therapy (WBRT) in patients with newly diagnosed primary central nervous system lymphoma. PATIENTS AND METHODS: Twenty-three patients were treated with HD-MTX on days 1 and 10. In case of at least a partial remission (PR), HD-BuTT followed by aPBSCT was given. Patients without response to induction or without complete remission (CR) after HD-BuTT received WBRT. RESULTS: Sixteen patients received HD-MTX and HD-BuTT achieving a CR/PR rate of 69%/13%. CR/PR rates for all patients (n = 23) were 70%/13%. There were three deaths during therapy. With longer follow-up three neurotoxic deaths occurred in irradiated patients (n = 9), while no persistent neurotoxicity was seen after HD-BuTT without subsequent WBRT. At a median follow-up of 15 months (range 1-69) median event-free survival (EFS) and overall survival (OS) for all patients were 17 and 20 months (Kaplan-Meier), after HD-BuTT 27 months and "not reached", respectively. Estimated 2-year EFS and OS were 45% and 48% for all patients versus 56% and 61% for the HD-BuTT group, respectively. CONCLUSION: MTX induction followed by HD-BuTT is an effective and very short time-on-treatment regimen. Median survival for patients treated with high-dose chemotherapy is not reached yet. The induction regimen needs optimisation. In this study WBRT was associated with a high incidence of severe neurotoxicity.

The modification of high-dose therapy shortens the duration of neutropaenia by delay of leucocyte nadir.

Infections during neutropaenia contribute still significantly to mortality and morbidity after high-dose therapy and autologous stem cell transplantation. Further acceleration of haemopoietic recovery seems impossible for biological reasons. Another approach to shorten neutropaenia could be to remove drugs from high-dose therapy protocols with strong contribution to immunosuppression and neutropaenia and unproven antineoplastic activity. In this retrospective matched-pair analysis, conventional busulphan/cyclophosphamide (Bu/Cy) high-dose therapy was compared to single-agent busulphan conditioning before autologous stem cell transplantation. This modification led to a significant shorter neutropaenic interval by protraction of cell decrease and to a significant mitigation of neutropaenia. After single-agent busulphan conditioning, leucocytes dropped below 1/nl at median 1.5 days later when compared to the patients from the busulphanBu/Cy control group (P=0.001). In a significant percentage of patients (n=6, 60%) leucocytes did not fall below 0.5 cells/nl at any time. In contrast, all patients from the Bu/Cy control group experienced deep neutropaenia (P=0.004). Thrombocytopaenia and requirement for transfusions of platelets or red cells were not influenced. Antineoplastic activity seemed to be preserved as determined by survival analysis. In conclusion, modification of high-dose regimen with the intention to shorten neutropaenia with preserved antitumour activity could be an approach to reduce infection-related morbidity and mortality and to consider economic necessities.

Antimicrobial prophylaxis in allogeneic bone marrow transplantation. Guidelines of the infectious diseases working party (AGIHO) of the german society of haematology and oncology.

Patients undergoing allogeneic stem cell transplantation are at high risk for infection with a variety of pathogens during different phases of the procedure. Bacteria and fungi predominate the first phase until engraftment. During the second phase, from engraftment to about day 100, major infectious problems are caused by fungi and cytomegalovirus. Both pathogens remain important under continued immunosuppression, however, in the late post-transplantation period infections with encapsulated bacteria may become a problem. In this review the Infectious Diseases Working Party of the DGHO gives recommendations for prophylaxis of infections under allogeneic stem cell transplantation with drugs and other measures. The aim of the group was to do this on an evidence-based-medicine rating, if possible.

Detection of disseminated epithelial cancer cells by liquid culture--factors interfering with the standardization of assays.

BACKGROUND: Immunocytochemistry is the standard method for detection of disseminated breast-cancer cells. Tumor-cell enrichment by cell culture has been used by several investigators, however assays published have not been well-standardized. METHODS: Breast-cancer cells from two lines were diluted in hemopoietic cells of varying origins and cultured in different media and different flasks. Factors influencing successful tumor-cell amplification by liquid culture were identified by investigation of 277 cultures. Parallel clinical samples, consisting of BM aspirations, leukapheresis samples and peripheral blood samples obtained from women with breast cancer, were investigated in 113 cultures. Cancer-cell detection by cell culture could be compared to immunocytochemistry in 101 cases. RESULTS: The frequency of tumor-cell detection was not improved by liquid culture, but a significant correlation between conventional tumor-cell detection and detection after liquid culture was found. Factors influencing tumor-cell amplification in the dilution assay could not be transferred to the investigation of clinical samples. It was concluded that culture-enrichment of disseminated cancer cells was very complex, and could be influenced by a variety of factors-even when a model system was used. DISCUSSION: It should be recognized that culture-enriched cancer cells probably represent a highly selected population of disseminated cancer cells, despite the significant correlation between tumor cells detected by conventional methods and following conventional methods after liquid culture. There is currently no evidence to suggest that cancer-cell amplification by cell culture could become a standardized technique for the detection of disseminated epithelial tumor cells.

Disseminated breast cancer cells prior to and after high-dose therapy.

Women with breast cancer in a distinct stage of disease can benefit from high-dose therapy (HDT) with autologous stem cell support; however, a significant number of these patients relapse despite this intensive treatment. This study investigates the persistence of malignancy on the single-cell level. A total of 194 data sets consisting of bone marrow and blood samples obtained prior to and after HDT and of aliquots of apheresis products were searched with immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) for disseminated cancer cells. Presence of cancer cells in the marrow is frequent prior to and after HDT, but HDT reduces the amount of malignant cells in marrow significantly. In contrast, there was no effect on the number of circulating cancer cells. Reinfusion of contaminated apheresis products was surprisingly associated with a low number of malignant cells in bone marrow after HDT and vice versa. The impact of disseminated tumor cells in bone marrow, apheresis, and peripheral blood on disease-free survival after HDT could be investigated in a total of 165 samples. Surprisingly, neither the presence of tumor cells in marrow or blood nor in apheresis was associated with a bad prognosis in Kaplan-Meyer survival analysis. These results suggest that apheresis products and bone marrow should be regarded as different biological compartments for epithelial cancer cells. It can be concluded that complete elimination of disseminated cancer cells by HDT is not always possible. The theory of reinduction of metastatic breast cancer by accidentally reinfused contaminants is not supported by this study so far. However, further research is necessary to identify distinct cell populations with the potentially capacity to metastasize.

Practices of infectious disease prevention and management during hematopoietic stem cell transplantation: a survey from the European group for blood and marrow transplantation.

Protocols for the prevention of infections after allogeneic or autologous hemopoietic stem cell transplantations are usual. A questionnaire was sent out to the members of the European Group for Bone and Marrow Transplantation (EBMT) in the spring of 1999. A total of 308 questionnaires from 180 centers were returned. Both allogeneic and autologous transplantation was reported from 128 centers, and allogeneic or autologous transplantation alone from four and 48 centers, respectively. Hemopoietic stem cell transplantation is still a domain of university hospitals. Intensive measures of isolation are usual. Allotransplantation is commonly performed in single rooms with HEPA-filtered air on special wards. However, even in the autologous setting, extensive measures of isolation are commonly used. This observation could be explained by historical developments and by the fact that nearly all centers for allogeneic transplantation perform both allogeneic and autologous transplantations, and thus similar measures are used in both settings. Other measures are usual but heterogeneous due to lack of clinical trials in this field. Drug prophylaxis during transplantation is mostly carried out with quinolones, TMP/SMZ, fluconazole, acyclovir, and pentamidine. Differences in drug prophylaxis after engraftment and in the use of different venous accesses do reflect the requirements after engraftment and discharge of patients from the transplant unit. The intensity of measures in autologous stem cell reinfusion does not reflect the development during the last decade. For cost effectiveness and convenience, it is necessary to abolish senseless measures. It is necessary to investigate anti-infectious strategies separately for allogeneic transplantation and other modalities of anti-cancer treatment in future.

Interference of cytokeratin-20 and mammaglobin-reverse-transcriptase polymerase chain assays designed for the detection of disseminated cancer cells.

Several reverse-transcriptase polymerase chain reaction (rtPCR) assays have been designed for the detection of disseminated cancer cells. The specificity of these discussed molecular approaches is controversial. Biological interference of the cytokeratin-20 and mammaglobin rtPCR assays has been investigated. Cell lines of different lineages and bone marrow and peripheral stem cells from patients without epithelial cancer have been examined for the transcription of the cytokeratin-20 (CK20) and mammaglobin messages prior to and after stimulation with different cytokines in a total of 370 liquid cultures. Amplification of both messages from clinical samples prior to stimulation does not support the high specificity for the detection of disseminated epithelial cancer cells as reported. Cytokeratin-20 was amplified from the chronic myeloic leukemia (CML)-derived line K562. Transcription was not influenced by cytokines, either in cell-line experiments or in clinical samples. The thesis of a low-level background transcription in granulocytes is supported. Mammaglobin was induced in cell lines significantly by GM-CSF and in clinical samples additionally by several more cytokines. These results indicate that under certain conditions involving cytokine production, the use of mammaglobin rtPCR for the detection of epithelial cancer cells could be limited. In conclusion, the mechanism of interference of both rtPCR assays are completely different and further research is necessary before the cytokeratin-20 or mammaglobin rtPCR could become standard methods for the detection of disseminated epithelial cancer cells. These factors leading to so-called false-positive results have to be considered in future applications of rtPCR for the detection of minimal residual disease.

ASHAP--an effective salvage therapy for recurrent and refractory malignant lymphomas.

BACKGROUND: This study was performed to examine the efficacy and toxicity of the combination of adriamycin (ADR), methylprednisolone (solumedrol), cytarabine (Ara-C), and cisplatin (CDDP) in patients with recurrent and refractory malignant lymphomas. PATIENTS AND METHODS: Sixty-five patients with Hodgkin's disease (HD) (n=14) or non-Hodgkin's lymphomas (NHL) (n = 51) were enrolled in the study. The ASHAP therapy consisted of ADR (40 mg/m2 by continuous infusion (CI) over 96 h), methylprednisolone (500 mg i.v., days 1-5), Ara-C (2 g/m2 as a 2-h infusion on day 5), and CDDP (100 mg/m2 by CI over 96 h). RESULTS: Twenty-five patients (38%) achieved complete remission (CR) and 20 (31%) were taken into partial remission (PR) for an overall response rate of 69%. Thirty-two patients with CR or PR following ASHAP underwent high-dose therapy (HDT) with subsequent hematopoietic stem cell transplantation. After a median follow-up of 52 months, 13 patients are in continuous CR (CCR), the 3-year event-free survival (EFS) was 30% for responders and 21% for all patients. The median overall survival (OS) was 12 months (range 0-70 months), and the OS rate after 3 years was 32%. Unfavorable prognostic factors for EFS and OS by univariate analysis were an elevated value of the serum lactate dehydrogenase and refractory lymphoma. The most frequently observed side effects following ASHAP were leukocytopenia and thrombocytopenia of World Health Organization (WHO) grades III/IV in approximately 80% of all courses. Non-hematological toxicities such as gastrointestinal side effects, infections, mucositis, renal and neurotoxicity occurred more rarely and reached WHO grades III/IV only occasionally. No treatment-related mortality with ASHAP was observed. CONCLUSIONS: ASHAP is an effective and moderately toxic salvage therapy for patients with recurrent or refractory HD and NHL. The results in patients responding to ASHAP and afterwards undergoing HDT with stem cell support are comparable with other established protocols and indicate an improvement in survival if HDT is carried out as intensification.

Treatment of mycotic infections after haemopoietic progenitor cell transplantation with liposomal amphotericin-B.

115 patients undergoing allogeneic or autologous bone marrow or peripheral blood stem cell transplantation were treated empirically or for documented fungal infection with liposomal amphotericin-B in doses up to 10mg/kg bodyweight for a duration up to 61 days. The therapy was excellent tolerated and clinical side effects occurred in only eight patients. The drug had to be withdrawn in one episode. A significant influence of liposomal amphotericin-B on laboratory parameters was not observed. Creatinine increased under therapy from a median base point of 1,0 (0,2-3,5) mg/dl to the upper normal value of 1,4 (0,4-4,2) mg/dl. Heavy increases of creatinine as well as of bilirubin, OT and PT were mostly associated with GvHD or regimen related toxicity. Considering the high-risk state of the patients the overall response rate was favourable with 62,9%. However, despite administration of liposomal amphotericin-B culture-proven mycoses were associated with a high morbidity (93,3%). Only one of fourteen patients was cured from Candida lambica septicaemia. We conclude that the antimycotic therapy with liposomal amphotericin-B has a low incidence of side effects. This should, considering the high mortality of fungal infections in BMT recipients, encourage investigators to perform dose escalating studies against the conventional formulation.

Nontransferrin-bound iron in serum of patients receiving bone marrow transplants.

Nontransferrin-bound iron (NTBI) and other parameters of iron status were measured in 40 patients undergoing bone marrow transplantation (BMT) prior to conditioning therapy (between day -10 and -7), at the time of BMT (day 0), and 2 weeks later (day + 14). Serum iron and transferrin saturation values were normal before conditioning therapy. At day 0 serum iron values were high and median transferrin saturation was 98% (changes in the values of both serum iron and transferrin saturation, p < .0001). Transferrin saturation values were still elevated 2 weeks posttransplant (day +14 vs. baseline values, p = .0001). Starting at low NTBI levels pretransplant (median 0.4 micromol/l, range 0-4.2 micromol/l, controls: < or = 0.4 micromol/l), all patients revealed high levels on day 0 (median 4.0 micromol/l, range 1.9-6.9 micromol/l, p < .0001) and 2 weeks posttransplant (median 2.7 micromol/l, range 0-6.2 micromol/l, p < .0001). These observations indicate that the plasma iron pool in patients undergoing BMT increases to a level at which the normal ability to sequestrate iron becomes exhausted and considerable amounts of NTBI appear in serum. This "free" form of iron can mediate the production of reactive oxygen species and may cause organ toxicity in the early posttransplantation period.

Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture.

The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation. Contaminations of autologous blood stem cells with cancer cells have been described. Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR. Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy. Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy. Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR. RNA was extracted by standard methods and reverse transcription was performed with MMV-RT. Integrity of RNA was checked by coamplification of housekeeping sequences. Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently. A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture. Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry. Ten leukaphereses from 6 women were available for PCR-analysis and cell culture. Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells. No sample was positive for cultured cells and negative for CK19-mRNA. Overall, the results corresponded in 80%. Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80%. PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary.

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes.

The small GTP-binding protein Rab3a is involved in regulated secretory pathways and is enriched in synaptic and neuroendocrine secretory vesicles. We have reported previously the developmental regulation of Rab3a in oligodendrocytes in culture and purified central nervous system myelin (Huber et al.: FEBS Lett 347: 273-278, 1994). Since multiple rab3 isoforms exist in the brain and may be associated with different secretory pathways, we have investigated the differential expression of the rab3 isoforms in oligodendrocytes, astrocytes, and Schwann cell line RT4-D6P2T. The expression of specific rab3 isoforms (rab3a-c) was detected by polymerase chain reaction (PCR) amplification and confirmed by sequence analyses. These data show that in addition to the previously reported expression in neurons, the two macroglial populations, astrocytes and oligodendrocytes, also express rab3 isoforms. Rab3b was preferentially amplified from purified, cultured astrocytes, while rab3a and rab3c were preferentially amplified from highly enriched populations of both cultured oligodendrocytes and those isolated directly from the brain by immunopanning. No novel rab3 isoform was detected in glia. These results indicate that glial cells in the brain express specific isoforms of the vesicular trafficking Rab3 protein family.

Inhibition of CFU-C growth by VP-16 containing plasma samples obtained from patients after conditioning therapy for bone marrow transplantation.

The introduction of VP-16 into high-dose therapy regimens used for conditioning before BMT or PBSCT has resulted in higher remission rates and prolonged disease-free survival, even in high risk patients. VP-16 levels have been measured in plasma at the time of transplantation. The question is, is there a biological activity that corresponds with the risk of delayed engraftment or graft failure? We investigated the inhibitory effects of plasma samples obtained from patients under high-dose VP-16 therapy on the growth of human bone marrow progenitor cells. Bone marrow cells from healthy donors were exposed to the plasma samples and seeded into methylcellulose-culture (CFU-C-assay). We found a dose dependent CFU-C inhibition related to VP-16 plasma levels at the time of transplantation (k = 0.769, P < 0.01). There were signs of a correlation between CFU-C growth inhibition at the time of BMT and haematological recovery (k = 0.656, P < 0.05) between CFU-C inhibition and the time until leucocytes reached 0.2 x 10(9)/l. Patients with CFU-C growth inhibition at the time of BMT may show delayed engraftment of leucocytes and that there might be a correlation with VP-16 levels, but further investigation is necessary to determine the significance of the latter thesis and if VP-16 plasma levels could lead to failure of engraftment. We recommend a minimum time interval between VP-16 infusion and graft transplantation of 72 h.