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Background Despite therapeutic approach that combines rituximab-containing chemotherapy, followed or not by autologous stem cell transplantation (auto-SCT), mantle cell lymphoma (MCL) patients experience relapses. Reduced-intensity conditioning allogeneic stem cell transplantation (RIC-allo-SCT) at time of relapse may represent an attractive strategy. Patients and methods We report a multicenter retrospective analysis. Seventy MCL patients underwent RIC-allo-SCT in 12 centers. Results Median age at transplantation was 56 years and median time from diagnosis to transplantation was 44 months. The median number of previous therapies was 2 (range, 1–5) including autologous transplantation in 47 cases. At time of transplantation, 35 patients were in complete remission, 20 were in partial response and 15 in stable disease or progressive disease. The median follow-up for living patients was 24 months. The 2-year event-free survival (EFS) and overall survival (OS) rates were 50% and 53%, respectively. The 1- and 2-year transplant-related mortality rates were 22% and 32%, respectively. The statistical analysis demonstrated that disease status at transplantation was the only parameter influencing EFS and OS. Conclusions These results suggest that RIC-allo-SCT may be an effective therapy in MCL patients with a chemo-sensitive disease at time of transplantation, irrespective of the number of lines of prior therapy. Studies are warranted to investigate the best type of RIC regimen.

The effectiveness of the novel hematopoietic stem cell mobilizing agent plerixafor was evaluated in nationwide compassionate use programs in 13 European countries. A total of 580 poor mobilizers with non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HL) and multiple myeloma (MM) were enrolled. All patients received plerixafor plus granulocyte CSF with or without chemotherapy. Overall, the collection yield was significantly higher in MM patients (>2.0 × 10(6) CD34+ cells/kg: 81.6%; >5.0 × 10(6) CD34+ cells/kg: 32.0%) than in NHL patients (>2.0 × 10(6) CD34+ cells/kg: 64.8%; >5.0 × 10(6) CD34+ cells/kg: 12.6%; P2.0 × 10(6) CD34+ cells/kg: 81.5%; >5.0 × 10(6) CD34+ cells/kg: 22.2%) than in NHL patients (P=0.013). In a subgroup analysis, there were no significant differences in mobilization success comparing patients with diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma. Our data emphasize the role of plerixafor in poor mobilizers, but further strategies to improve the apheresis yield especially in patients with NHL are required.

Donor KIR haplotype B improves progression-free and overall survival after allogeneic hematopoietic stem cell transplantation for multiple myeloma

Contains fulltext : 96501.pdf (Publisher’s version ) (Closed access) The aim of our study was to evaluate, through the Eurocord and European Group for Blood and Marrow Transplantation (EBMT) registries, outcomes and risk factors for outcomes in adult patients who underwent single or double unrelated cord blood transplantation (UCBT) for myelodysplastic syndrome (MDS) or secondary acute myeloblastic leukemia (sAML). A total of 180 adults with MDS (n=39) or sAML (n=69) were analyzed. Risk factors for outcomes were analyzed using the Fine and Gray method and the Cox model. Median age was 43 (18-72) years. In all, 77 patients (71%) received a single UCBT. Myeloablative conditioning regimen (MAC) was given to 57 (53%) patients. Median numbers of nucleated and CD34(+) cells at freezing were 3.6 x 10(7) and 1.1 x 10(5) kg. At 60 days, cumulative incidence of neutrophil recovery was 78+/-4% and was independently associated with the number of CD34(+) cells per kg (>1.1 x 10(5); P=0.005) and advanced disease status (blasts 5% and International Prognostic scoring system (IPSS) intermediate-2 or high in MDS) had significant poorer DFS (hazard ratio (HR): 1.76; P=0.047). In spite of high NRM, these data indicate that UCBT is an acceptable alternative option to treat adults with high-risk MDS or sAML, without a suitable human leukocyte antigen (HLA)-matched donor.

The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2-75)) were given 240 μg/kg plerixafor SC 9-11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 10(6) CD34(+) cells/μL. The median cell yield was 3.35 × 10(6) CD34+ cells/kg (0-29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 10(6) CD34+ cells/kg (1.6-5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 10(6) CD34+ cells/kg (0-24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.

Monitoring of minimal residual disease (MRD) after allogeneic (allo)-SCT for myelofibrosis (MF) allows recognizing the depth of remission and thus guides application of appropriate therapeutic interventions. MPL W515L/K mutations, which are detected in 5-10% of JAK2V617F-negative patients, may be useful for this purpose. Using a highly sensitive quantitative PCR method, we tested 90 patients with MF who underwent allo-SCT for the presence of MPL W515L/K mutations. Two patients with primary MF were found to harbor MPLW515L while no patient was positive for MPLW515K mutation. Both patients were JAK2V617F negative and cleared the mutation rapidly after allo-SCT and remained negative for a median follow-up of 19 months. The results of molecular monitoring correlated well with other remission parameters such as normalization of peripheral blood counts and morphology and complete donor chimerism. We conclude that MPLW515L can be cleared after allo-SCT and hence may be used as an MRD marker in a proportion of JAK2V617F-negative MF patients.

Patients with AML or myelodysplastic syndrome who relapse after allo-SCT have a poor prognosis. In the search for novel treatment strategies for these patients, we conducted a multicenter retrospective analysis and identified 22 patients treated with the DNA-methylation inhibitor 5-azacytidine (5-Aza). Patients received a median number of two cycles 5-Aza (range 1-8) at a dose of 100 mg/m(2) over 5 days following relapse. Eighteen patients (82%) also received a median number of two donor lymphocyte infusions (DLI, range 1-5). Sixteen patients (72%) responded to 5-Aza treatment and five patients (23%) achieved a CR. 5-Aza-induced CR lasted for 433 days (median, range 114-769). Median survival and the estimated 2-year survival rate were 144 days and 23%, respectively. Acute GVHD after DLI was seen in six patients (33%) and four of these patients developed chronic GVHD of the skin. There were no treatment-related deaths. Patients who achieved halving of leukocyte counts after the first 5-Aza cycle had a superior median survival of 802 days compared with 135 days (P=0.0025) in all other patients. On univariate analysis, the achievement of this halving of leukocyte counts was identified as a significant predictor of survival.

Contaminating breast cancer cells in leukapheresis harvested for reinfusion to rebuild hemopoiesis after high-dose therapy have been described by several investigators. Methods for tumor cell detection are conventional immunocytochemistry, culture techniques and reverse transcriptase PCR. The percentage of tumor cell positive leukaphereses shows a wide variation. An approach to clarify if these cells can induce systemic relapse is to characterize them molecular-genetically and immunologically, but these techniques require a sufficient cell count.We compared conventional immunocytochemistry with immunocytochemistry after immunomagnetic enrichment of cancer cells by HEA-125 magnetic microbeads for the detection of micrometastatic tumor cells. A total of 25 samples, consisting of 16 samples from G-CSF-mobilized peripheral stem cell harvests, eight BM aspirations and one peripheral blood sample were investigated without [median 2, range 1-3 x 10(6) MNCs] and after [median 5, range 1-10 x 10(7) MNCs] HEA-microbead selection. Additionally 10 buffy coat samples from healthy subjects were investigated.Using conventional immunocytochemistry, tumor cells could be detected in nine stem cell samples. Two BM samples and the blood sample (48%) were positive, with a median tumor cell load of 0 (0-12) cells per sample (mean: 2.4). By HEA-bead selection the rate of positivity could be increased to 88% (13 stem cell samples, eight marrow samples and one blood sample) with a median load of 6 (0-47) (mean 10.6) suspected cells (p0.007). However, calculation of recovery revealed tumor cell losses by immunobead selection. False positive results were not seen.We conclude first that immunomagnetic selection is an excellent and highly sensitive tool to enrich contaminating cancer cells from marrow and stem cell samples; second that the existence of real tumor cell negative stem cell harvests is doubtful; and third that immunobead selection delivers sufficient tumor cell counts for their further characterization by molecular and immunological methods.

Allogeneic SCT is important in myelodysplastic syndrome, the BCR-ABL-negative chronic myeloproliferative diseases (CMPDs) and in poor-risk AML. Techniques to monitor the minimal residual disease, for example, by PCR or immunophenotyping gain increasing importance in the post transplantation period as basis for improved and earlier therapeutic interventions in impending relapse. Recent markers such as the NPM1 mutations in AML or the JAK2V617F mutation in the CMPD can be exactly quantified by real-time PCR and were evaluated for their prognostic value in the post transplantation phase and for their utility to plan adoptive immunotherapy in case of molecular relapse. With respect to chimerism, new and very sensitive methods were introduced, for example, quantitative assessment of genetic polymorphisms by real-time PCR, but also methods here are still highly individualized. Only in CML, where SCT focuses now on poor-risk cases or cases of tyrosine kinase inhibitor failure, follow-up schedules are standardized. Standardization of the different diagnostic techniques and of the intervals in the post transplantation period is urgently needed also in other myeloid malignancies and should be focus of future studies.

We analyzed the prognostic impact of the most frequent genetic abnormalities detected by fluorescence in situ hybridization in 101 patients with multiple myeloma, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) after melphalan/fludarabine-based reduced conditioning. The incidences of abnormalities in the present analysis were as follows: del(13q14) (61%), t(11;14)(q13;q32) (14%), t(4;14)(p16.3;q32) (19%), MYC-gain gains (8q24) (21%), del(17p13) (16%) and t(14;16)(q32;q23) (5%). None of the patients had t(6;14)(p25;q32). The overall complete remission (CR) rate was 50% with no differences between the genetic abnormalities except for patients with del(17p13) who achieved less CR (7 vs 56%; P=0.001). Univariate analysis revealed a higher relapse rate in patients aged >50 years (P=0.002), patients with del(13q14) (P=0.006) and patients with del(17p13) (P=0.003). In multivariate analyses, only del(13q14) (HR: 2.34, P=0.03) and del(17p13) (HR: 2.24; P=0.04) significantly influenced the incidence of relapse, whereas for event-free survival, only age (HR 2.8; P=0.01) and del(17p13) (HR: 2.05; P=0.03) retained their negative prognostic value. These data show that del(17p13) is a negative prognostic factor for achieving CR as well as for event-free survival after HSCT. Translocation t(4;14) might be overcome by allogeneic HSCT, which will have implication for risk-adapted strategies.

Investigation of high-dose chemotherapy (HD-CT) compared with standard-dose chemotherapy (SD-CT) as adjuvant treatment in patients with primary breast cancer and >/=10 axillary lymph nodes. From November 1993 to September 2000, 307 patients were randomized to receive after four cycles of epirubicin (90 mg/m(2)), cyclophosphamide (600 mg/m(2)) i.v. (every 21 days) and either HD-CT of cyclophosphamide (1500 mg/m(2)), thiotepa (150 mg/m(2)) and mitoxantrone (10 mg/m(2)) i.v. for four consecutive days followed by stem cell transplantation or a SD-CT of three cycles CMF (cyclophosphamide 500 mg/m(2), methotrexate 40 mg/m(2), 5-fluorouracil 600 mg/m(2), i.v. on day 1 and 8, respectively, every 28 days). After a median follow-up of 6.1 years, 166 events with respect to event-free survival (EFS) (SD-CT: 91, HD-CT: 75) have been observed. The hazard ratio of HD-CT versus SD-CT is estimated as 0.80 [95% confidence interval (0.59, 1.08)], P = 0.15. The trend to a superiority of HD-CT as compared with SD-CT with respect to EFS seems to be more pronounced in premenopausal patients as compared with postmenopausal patients and in patients with tumor grade 3 as compared with patients with tumor grade 1/2. With a follow-up of 6 years, there was a trend in favor of HD-CT with respect to EFS not being significant. A proper meta-analysis needs to be undertaken for an evaluation of subgroups of patients who might benefit from HD-CT.

Optimal clinical management of patients with primary myelofibrosis and post-essential thrombocythemia/polycythemia vera myelofibrosis is a challenge, given the typically advanced age of presentation and variability of the disease course and prognosis. Current medical therapeutic options have not demonstrated an impact on the disease course, which exceeds the palliation of disease-related extramedullary hematopoiesis and alleviation of cytopenias. In contrast, allogeneic stem cell transplantation (SCT) can lead to 'cure' but is limited due to patient's age or comorbidities. Currently, in patients, who are reasonable candidates, SCT (frequently with a reduced intensity conditioning regimen) is employed for intermediate- to high-risk disease. Current pharmaco-medical therapy is used as a bridge to transplant, or instead of transplant in poor transplant candidates. Pathogenetic insights, especially the discovery of the Janus kinase (JAK)2(V617F) mutation, have ushered in a host of new potential therapeutic agents that may augment the role of medical therapy. Similarly, the boundaries of transplantation continue to alter with strategies that decrease conditioning-related toxicity, improved antimicrobial prophylaxis and decreased graft-versus-host disease. The potential for continued improvements in both medical and transplant therapy suggests that for the immediate future the optimal choices for an individual patient will remain potentially volatile and present complex decisions.

Scant knowledge exists concerning lineage-restricted mixed chimerism (mCh) after allogeneic peripheral blood stem cell transplantation (PSCT) in patients with chronic idiopathic myelofibrosis (CIMF). Following a sex-mismatched PSCT, a combined immunopheno- and genotyping by fluorescence in-situ hybridization (FISH) was performed on sequential bone marrow (BM) biopsies at standardized intervals. Results were compared with PCR analysis of corresponding peripheral blood samples in five patients. According to FISH, pretransplant specimens revealed a gender congruence of more than 99%, while in the first three months the total BM exhibited a persistent fraction of host cells (30% to 40%) with a tendency to decline after about one year. It is noteworthy that the majority of endothelial cells maintained a recipient origin, whereas CD34+ progenitors and especially CD61+ megakaryocytes exhibited only very few host-derived cells. In keeping with the prevalence of donor cells in the hematopoietic compartment, PCR analysis of peripheral blood cells displayed a non-significant degree of mCh. In conclusion, according to FISH and PCR analysis, successful PSCT in CIMF results in an almost complete chimeric (donor-derived) state of the hematopoietic cell population. The non-transplantable stromal compartment includes the vascular endothelium with a predominance of recipient cells. The minimal mCh of this population implies probably a donor-derived origin (endothelial progenitor cells).

Multiple myeloma (MM) is the second most common hematological malignancy and develops from a malignant plasma cell (PC) clone within the human bone marrow (BM). The proliferation and expansion of the malignant PC producing a monoclonal immunoglobulin eventually results in BM failure with anemia, skeletal involvement with osteolytic lesions and hypercalcemia, and renal failure due to the excessive production of paraprotein.1

In advanced stage mantle cell lymphoma, conventional chemotherapy yields a complete remission rate below 40%, and the median survival rate is only about 3 years. Between 1991 and 1996 we treated nine such patients (six male; three female) with high-dose chemotherapy (six of these also with 12 Gy fractionated total body irradiation (TBI)) and peripheral stem cell support (n = 8) or allogeneic bone marrow transplantation (n = 1). The median age was 47 years (range, 28-61). At the time of high-dose chemotherapy, five patients were in first complete remission (CR), two in first partial remission (PR) and two in second remission (CR = 1; PR = 1). High-dose chemotherapy included TBI (12 Gy), etoposide and cyclophosphamide (patients 1-5), TBI and cyclophosphamide (patient 7), busulfan, etoposide and cyclophosphamide (patients 6 and 9), cyclophosphamide and busulfan (patient 8). The patterns of toxicity according to the Bearman score were usually mild (mucositis grade 2, n = 7; renal grade I, n = 2) with no therapy-related fatality. Only one patient developed hepatic toxicity grade III (veno-occlusive disease) but recovered completely. The median time to neutrophil engraftment was 10 days (range, 8-15). After high-dose chemotherapy all patients achieved complete remission. After a median follow-up of 22 months (range, 9.4-64) all patients remain in continuous complete remission. These encouraging results suggest that high-dose chemotherapy can be applied safely and leads to long-term disease-free survival in otherwise incurable disease.

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH), hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon, we investigated the usefulness of ALDH activity for characterizing HSC graft quality, particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time, and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly, in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells, this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism, whereas all other patients showed early complete donor chimerism. In conclusion, we suggest to measure ALDH activity as a surrogate marker for HSC activity, and to transport and store PBSC under controlled cooling conditions.

Contains fulltext : 33189schattenberg.pdf (Publisher’s version ) (Closed access) The impact of the donor gender on outcome in HLA-identical sibling donor hematopoietic stem cell transplantation for multiple myeloma was studied in a retrospective registry study of 1312 patients (476 male to male (M --> M); 334 female to male (F --> M); 258 male to female (M --> F); 244 female to female (F --> F) reported to the European Group for Blood and Marrow Transplantation (EBMT). The best overall survival (OS) from the time of transplantation was found in F --> F (median 41 months) with no significant difference between other groups (median 25 months in M --> M, 18 months in F --> M, 19 months in M --> F) despite a significantly higher nonrelapse mortality in F --> M. This was due to a significantly lower relapse rate (REL) in F --> M compared to all other groups. Before 1994, OS was poorer in F --> M than in M --> M, which improved to similarity from 1994 onwards (median 29 months in M --> M and 25 months in F --> M). The reduced REL contributed to this improvement in F --> M indicting a gender-specific graft vs myeloma effect. Therefore, a female donor is as good as a male one for male patients, while for female patients gender disparity is a negative factor for outcome.

Allogeneic stem cell transplantation (SCT) is a potentially curative approach for patients with hematological malignancies. Reduced-intensity conditioning regimens allow SCT in elderly patients; however, there are only limited data on the feasibility and outcomes of unrelated donor SCT in these patients. In this study, we analyzed, retrospectively, data of 36 patients with various hematological malignancies and median age 58 years (range, 55-66), who were given unrelated donor SCT after reduced-intensity conditioning. The preparative regimen consisted of fludarabine combined with oral busulfan (8 mg/kg, n=8), intravenous busulfan (6.4 mg/kg, n=11), treosulfan (30 g/m(2), n=5) or melphalan (100-150 mg/m(2), n=12). Patients were also given serotherapy, ATG (n=32), or alemtuzumab (n=4). The probabilities of overall survival, disease-free survival, and nonrelapse mortality at 1 year after SCT were 52, 43, and 39%, respectively. Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD occurred in 31 and 45%, respectively. Multivariable analysis determined that survival rates were higher in patients with chemosensitive disease (HR 4.5), and patients conditioned with intravenous busulfan or treosulfan (HR 3.9). Unrelated donor SCT is feasible in elderly patients, with outcomes that are similar to younger patients. Favorable outcome was observed in patients with myeloid malignancies, and those transplanted in remission and early in the course of disease. Age alone should not be considered a contraindication to unrelated donor SCT.

Allogeneic stem cell transplantation is an established treatment modality for a variety of hematologic malignancies. However this allogeneic stem cell transplantation carries the risk of severe complications which are partly due to the intensity of the conditioning regimen. Therefore, allogeneic stem cell transplantation has been so far only performed in younger patients with good performance status. Dose-reduced or non-myeloablative conditioning regimen has been proposed as an alternative that would allow exploiting the graft versus leukemia effect of allogeneic stem cell transplantation without severe toxicity. Clinical studies have shown that dose-reduced conditioning followed by allogeneic stem cell transplantation allows stable engraftment with lower toxicities which can be associated with long-term disease control. Therefore, dose-reduced conditioning followed by allogeneic stem cell transplantation can be offered to older and more debilitated patients who were no candidates for standard allogeneic stem cell transplantation.

It is unknown whether the addition of antithymocyte globulin (ATG) to reduced-intensity conditioning with busulfan (BU) and fludarabine (FLU) is beneficial in HLA-identical sibling transplantation. Therefore, we analyzed retrospectively data on 83 patients, who received peripheral blood stem cells from HLA-identical siblings after conditioning with either 8 mg/kg BU and 150 mg/m2 FLU (n=45) or 8 mg/kg BU, 180 mg/m2 FLU and 40 mg/kg ATG (n=38). Graft-versus-host disease (GVHD) prophylaxis consisted of CSA alone (n=32) or a combination with either MTX or MMF (n=51). The median age was 52 years. Graft failure occurred in two patients after BU/FLU and in three after BU/FLU/ATG (P=0.66). After conditioning with BU/FLU, platelet recovery was significantly faster (P=0.017), and less platelet (P

We assessed the effect of rabbit antithymocyte globulin manufactured by Fresenius (ATG-F) on the hemostatic system in patients (n=12) with various hematological malignancies undergoing hematopoietic stem cell transplantation (HSCT) from HLA-matched unrelated donors. For this purpose, we monitored different parameters of coagulation before, during and after the administration of ATG-F. As a control group, we recruited patients (n=10) undergoing HSCT from their HLA-identical siblings who did not receive ATG-F as part of their preparative regimens. At 24 and 48 h after ATG-F treatment had been initiated, we found a temporary rise in D-Dimer, tissue factor, soluble thrombomodulin and thrombin-antithrombin III complex levels and a significant decrease of platelet counts in patients treated with ATG-F as compared to the control group. No differences between the two groups could be detected with regard to global coagulation tests as well as the incidence of bleeding manifestations, thromboembolic complications or the development of vascular-occlusive-disease of the liver. This temporary state of a stressed but compensated coagulation system under ATG-F therapy can be addressed as nonovert disseminated intravascular coagulation (DIC). The effect was independent from the different conditioning regimens and eased off after cessation of ATG-F. We conclude that ATG-F can induce nonovert DIC in patients receiving antithymocyte globulin as part of their conditioning regimen for HSCT.

To evaluate the schedule dependency of G-CSF (filgrastim) for stem cell mobilisation we conducted a randomized trial in 50 healthy donors, with one subcutaneous daily injection of 10μg/kg G-CSF (n=25) compared to twice injections daily of 5μ/kg G-CSF (n=25). The two groups were well balanced for age, bodyweight, and gender. G-CSF application was performed on an out-patient basis and leukapheresis was started in all donors on day 5. The most frequent side effects of G-CSF were mild to moderate bone pain (88%), mild headache (72%), mild fatigue (60 and 48%) and nausea (8%) without differences between the two groups.

To reduce the incidence of severe graft versus host disease (GvHD) and lower the treatment related mortality(TRM) in allogeneic stem cell transplantation from HLA-identical siblings patients with multiple myeloma, we incorporated anti-thymocyte globulin (ATG, Fa Fresenius, Bad Homburg, Germany) in the conditioning regimen of 12 patients. The conditioning regimen consisted of modified total body irradiation, busulfan and cyclophoshamide (n=9) or busulfan and cyclophoshamide (n=3). The median age was 44 years (range, 29–53) and the median time from diagnosis to transplant was 12 months (range, 6 to 56). The stem cell source was bone marrow in 10, and peripheral blood stem cells in 2 patients. Grade II-IV acute GvHD occurred in 3 patients (27% percent). Severe grade III and IV GvHD developed in only 1 patient. Major toxicity was mucositis. Grad II according the Bearman score was noted in 10 patients, whereas 2 patients experienced grade III, requiring prophylactic intubation. One patient died of severe GvHD grade IV and one patient developed multi-organ failure on day +13, resulting in a TRM of 17%. A complete response in surviving patients after allogeneic transplantation was seen in 4 (40%), and PR in 6 (60%) patients. Two of the patients with PR received a donor lymphocyte infusion (DLI) for further tumor reduction 8 and 14 months after stem cell transplantation, and converted to CR, which increased the rate of CR up to 60%. After a median follow-up of 25 months (range, 5–62), no patient with CR after allogeneic transplantation relapsed during follow-up, while 3 out of 4 patients with PR. experienced progress within 3 years (p=0.07). The estimated overall survival at three years for all patients is 83 percent (CI 95%: 68%–98%). The estimated progression-free survival at three years is 61 percent (CI 95%: 32%–90%). These results suggest that the incorporation of anti-thymocyte globulin may prevent severe GvHD without obvious increase of relapse. DLI should be administered for patients with incomplete response after transplantation to enhance the rate of complete remission, which results in long term freedom of disease.

Unrelated donor (UD) hematopoietic stem cell transplantation (HSCT) is accepted as a therapy for leukaemic diseases and varying inborn diseases if a suitable related donor cannot be found. The goal of immunosuppressive therapy with UD-HSCT is an effective prevention of graft-versus-host-disease (GvHD) on one hand. On the other hand an optimal balance with immunocompetence of the transplanted bone marrow is desirable in order to prevent graft failure, infection and, in the case of leukaemic diseases, potentially control the underlying disease.Between 1992 and 2000 49 patients aged 11 months to 16.7 years received an UD-HSCT in Hamburg. Underlying diseases were leukaemia or MDS in 35, of these ALL in 21, hemophagocytic lymphohistiocytosis (HLH) in 9, immunodeficiency or inborn error of metabolism in 5 patients. GvHD-prophylaxis consisted of a combination of Cyclosporin A (CSA), methotrexate (MTX), metronidazole, IgM-enriched iv-immunoglobulin (ivIg) (Pentaglobin(R)) or ivIgG and anti-thymocyte-globulin (ATG). Within the same time span 10 patients with ALL received a matched related donor HSCT (MRD-HSCT). GvHD-prophylaxis in these patients was done without ATG in 8 of 10 cases. UD-HSCT were analyzed for survival, relapse and toxicity. Probability of survival of the patients with ALL after UD-HSCT was compared with results of MRD-HSCT in children with ALL.The Kaplan-Meier estimates of three year overall-survival (OS) were 74 % for all patients. Probability of disease-free survival (DFS) at three years was 62 % for leukaemia/MDS-patients and 100 % for the HLH-patients. Acute GvHD (aGvHD) grades II or III occurred in 51 % of patients. Chronic GvHD (cGvHD) occurred in 22 % of patients. There were 5 cases of treatment-related mortality (TRM). Probability of DFS for patients with ALL at three years was 65 % after UD-HSCT and 30 % in the patients after MRD-HSCT.UD-HSCT in children is an effective and safe therapy. A GvHD-prophylaxis regimen combining the standard immunosuppressive agents CSA and MTX with ivIg, metronidazole and serotherapy using ATG may result in a low incidence of severe GvHD-complications and low TRM rate without increase in relapse rates.

Chronic myeloid leukemia in blast crisis (BC) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) are associated with extremely poor outcome. Allogeneic transplantation during BC or active leukemia is most often unsuccessful due to high-rates of both treatment-related complications and relapse. Long-term results are significantly better if a second chronic phase or remission can be achieved prior to transplantation. Similarly, DLI given for the treatment of post-transplant relapse is more successful when given during a second remission. In this study we report our results with a previously unreported approach consisting of short-term treatment with imatinib mesylate (formerly, STI571) to induce or maintain remission, followed by allogeneic transplantation or DLI and the impact on transplantation/DLI outcome. Sixteen patients were treated either in preparation for transplantation (n = 12), for DLI (n = 1), or for both (n = 3). Ten had CML in BC; seven myeloid and three lymphoid BC. Six patients had Ph(+) ALL. The donors were matched unrelated (n = 9), matched siblings (n = 5) or haplo-identical (n = 2). Eleven of 15 patients given imatinib pre-transplant were transplanted in complete hematologic response. Engraftment and GVHD rates were not different from expected. Seven patients had grade II-III hepatic toxicity after transplantation. After a median follow-up of 10 months (range, 3-16 months) six remain alive, two after further therapy. The 1-year survival rate was 25%. Four patients were given imatinib prior to DLI, all had complete response. Two remain in remission >6 months from relapse. In conclusion, treatment with imatinib allows transplantation in a more favorable status or maintaining remission with low toxicity until transplantation is feasible. Pre-transplant imatinib seems safe and not associated with excess post-transplant complications. Imatinib may have substantial activity in combination with DLI. Further study of a larger group of patients is required to assess the impact on long-term outcome and the role of post-transplant imatinib in controlling residual disease.

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.

Cancer-testis antigen MAGE-C2/CT10 induces spontaneous CD4 + and CD8 + T-cell responses in multiple myeloma patients

This retrospective study describes the outcome of patients with chronic myeloid leukaemia after allografting using dose-reduced conditioning with fludarabine and busulphan. Forty-four Philadelphia chromosome (Ph)-positive patients were transplanted in nine German centres; 26 patients were in chronic phase, 11 in accelerated phase and seven in blast crisis. Thirty-four patients achieved complete remission, with 18 alive and disease-free at a median follow-up of 562 d (range 244-922 d). Grade II-IV acute graft-versus-host disease (GVHD) incidence was 43%. Twenty patients died, 15 of causes unrelated to relapse. Risk factors predisposing to graft failure by univariate analysis were an unrelated donor (8/23 compared with a related donor 2/21, P = 0.07) and interferon therapy within 90 d of transplant (4/6 versus 3/17, P = 0.025). At the last follow-up, of 31 patients for whom molecular or cytogenetic data were available, 16 (52%) were polymerase chain reaction-negative, and seven (23%) were Ph-negative by fluorescent in situ hybridization. These findings demonstrate that dose-reduced conditioning with fludarabine and busulphan provides durable engraftment and a low rate of relapse. However, in this population, many of whom were not eligible for high-dose conditioning due to age, reduced performance status, previous complications or extensive pre-treatment, these data highlight the need for effective anti-infectious and GVHD prophylaxis. In addition, this study supports the discontinuation of interferon therapy at least 90 d before transplant

Two silica-precipitating peptides, silaffin-1A(1) and-1A(2), both encoded by the sil1 gene from the diatom Cylindrotheca fusiformis, were extracted from cell walls and purified to homogeneity. The chemical structures were determined by protein chemical methods combined with mass spectrometry. Silaffin-1A(1) and -1A(2) consist of 15 and 18 amino acid residues, respectively. Each peptide contains a total of four lysine residues, which are all found to be post-translationally modified. In silaffin-1A(2) the lysine residues are clustered in two pairs in which the epsilon-amino group of the first residue is linked to a linear polyamine consisting of 5 to 11 N-methylated propylamine units, whereas the second lysine is converted to epsilon-N,N-dimethyllysine. Silaffin-1A(1) contains only a single lysine pair exhibiting the same structural features. One of the two remaining lysine residues was identified as epsilon-N,N,N-trimethyl-delta-hydroxylysine, a lysine derivative containing a quaternary ammonium group. The fourth lysine residue again is linked to a long-chain polyamine. Silaffin-1A(1) is the first peptide shown to contain epsilon-N,N,N-trimethyl-delta-hydroxylysine. In vitro, both peptides precipitate silica nanospheres within seconds when added to a monosilicic acid solution.

We investigated the efficacy and toxicity of the combination of busulfan, cyclophosphamide, and etoposide (Bu/Cy/VP-16) as a preparative regimen prior to autologous hematopoietic stem cell transplantation (ASCT) in patients with Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL). Fifty-three patients with recurrent ( n=30), refractory ( n=20), or high-risk ( n=3) lymphoma were enrolled. The 10 patients with HD and 43 with NHL (median age: 46 years, range: 18-64) received busulfan (16 mg/kg), cyclophosphamide (120 mg/kg), and etoposide (30 or 45 mg/kg) followed by ASCT. A total of 50 patients (94%) were consolidated in complete ( n=25) or partial ( n=25) remission, whereas 3 patients had chemoresistant disease before Bu/Cy/VP-16. Thirty-five patients (66%) had received prior radiotherapy (RT) excluding total body irradiation (TBI) as part of the conditioning regimen. The main nonhematological toxicities (grade II-IV according to the Bearman score) in 52 evaluable patients were mucositis (79%) and hepatic toxicity (15%). Severe veno-occlusive disease (VOD) occurred in three patients (5.8%) including one treatment-related death caused by VOD. Overall, treatment-related mortality was 3.8%. After a median follow-up for surviving patients of 21 months (range: 6-118), 20 patients (38%) are in continuous complete remission, 8 patients (15%) are alive in relapse, and 25 patients (47%) died. Probabilities of relapse, event-free survival, and overall survival at 3 years were 63% [95% confidence interval (CI): 48-79%], 31% (95% CI: 17-46%), and 43% (95% CI: 27-59%), respectively. In conclusion, Bu/Cy/VP-16 is an effective and well-tolerated conditioning regimen in patients with HD and NHL. Both toxicity and outcome were not significantly different in patients treated with 30 mg/kg and 45 mg/kg etoposide, respectively. The observed long-term results are even comparable to those published for other established high-dose protocols, including TBI-based regimens. However, further investigations are necessary to evaluate the value of Bu/Cy/VP-16 as a high-dose protocol for malignant lymphoma, especially in patients who have already received extensive RT.

We report about a 28-year-old woman with relapsed mantle cell lymphoma (MCL, centrocytic lymphoma according to the Kiel classification) refractory to salvage chemotherapy. The patient underwent allogeneic bone marrow transplantation from a HLA-identical brother after myeloablative chemotherapy consisting of busulfan, etoposide, and cyclophosphamide. The patient experienced hepatic toxicity (grade I), mucositis (grade II) according the Bearman scale, and graft versus host disease of the skin (grade II) and showed stable engraftment with complete chimerism on day 15 after bone marrow transplantation. Eight years after transplantation, the patient is still disease free and in good condition without any late side effects. This report suggests a curative potential of allogeneic stem cell transplantation in MCL.

Lineage-specific gene expression has been used for the identification of metastasis of cancers with unknown primary site or of disseminated cancer cells in haemopoietic compartments such as bone marrow or in lymph nodes. For the muc1, cytokeratin-19 and the CEA genes, the transcription in haemopoietic cells has been shown recently. Here, the expression of the mammary epithelium related antigens BA46 (lactadherin) and BA70 in lymphoid and myeloid cell lines, and in clinical specimens is analysed. By Northern-hybridization with specific oligonucleotides an ubiquitous transcription of both genes, independent from the provenance of cells or the chromosomal gender was found. Both mRNA molecules were amplified by rtPCR from the samples and the specificity could be confirmed by sequence analysis. Peptide-specific antibodies were raised in rabbits and used for Western-blot analysis and for immunocytochemical studies. Both antibodies reacted with total cell lysates from myeloid and lymphatic cells. In immunocytochemistry antibody P717 (anti-lactadherin) had a significant strong staining of the myeloid cell lines K562 and HL60 suggesting a participation of lactadherin in leukocyte-function. Using antibody P718, strong stains were seen in myeloid line K562 and lymphoid line ST486. In conclusion, our findings expand the results that the concept of lineage-specific gene expression is no longer valid at the molecular level. © 2000 Cancer Research Campaign

We compared fractionated total body irradiation (12 Gy)/cyclophosphamide (120 mg/kg) with busulfan (16 mg/kg)/cyclophosphamide (120 mg/kg) as preparative therapy in unrelated donor stem cell transplantation of CML patients. Fifty patients with CML (1.CP = 46; aP = 4) and a median age of 36 years (range 16-52) were enrolled in this sequential trial between 1994 and 1999. In both groups patients were well balanced with respect to age, disease status, stem cell source and CMV status. All patients received standard doses of cyclosporin A, methotrexate and anti-thymocyte globulin (ATG) as GVHD prophylaxis. No graft failures occurred in either group. The median day of leukocyte engraftment was earlier in the Bu/Cy than in the TBI/Cy group (day 15 vs 17; P = 0.006). The incidence of grade II-IV GVHD was 40% in the TBI/Cy and 36% in the Bu/Cy group, whereas severe grade III/IV GVHD was only observed in 12% of patients in both groups. The incidence of chronic GVHD (limited and extensive) at 1 year was higher in the Bu/Cy arm (65% vs 30%; P = 0.02). More toxicity grade I/II of the liver (88% vs 44%; P = 0.002) and more hemorrhagic cystitis (32% vs 8%; P = 0.02) were observed in the Bu/Cy regimen. Seven relapses in the TBI and no relapse in the Bu/Cy group were observed after a median follow-up of 44 and 15 months, respectively. The estimated 3 year OS and DFS was 72% (95% CI: 55-98%) and 58% (95% CI: 39-77%) in the TBI and 70% (95% CI: 51-89%) for DFS and OS in the Bu/Cy group. We conclude that the anti-leukemic effect of the Bu/Cy regimen seems to be at least as effective as the TBI/Cy combination in unrelated stem cell transplantation of CML patients, with no graft failures, but that it correlates with a higher incidence of liver toxicity, hemorrhagic cystitis and chronic GVHD. Longer follow-up is necessary to determine the late relapse rate and late toxicity.

Mit medianen Uberlebenszeiten von etwa 2 Jahren gilt das metastasierte Mammakarzinom als inkurables Erkrankungsstadium. Da z. B. selbst nach antrazyklinhaltiger Polychemotherapie die Rate der rezidivfrei Uberlebenden nach 5 Jahren bei 3,1% liegt [1], gilt derzeit allgemein die Behandlungsdevise: nihil nocere.

Breast cancer is a chemosensitive tumour and anthracyclines are one of the most active cytotoxic agents in chemotherapy treatment. Failure after anthracycline-containing chemotherapy is a poor prognostic factor because of low response rate to salvage chemotherapy. Several factors like P-glycoprotein mediated drug resistance (MDR-1 or MRP), glutathione or amplification of topoisomerase II have been found to be involved in anthracycline resistance. No clear benefit for patients treated with 'resistance-modifier' agents like verapamil, dexverapamil or quinidine has yet been demonstrated. Most clinical studies with non-cross resistant cytotoxic agents are lacking a strict definition of anthracycline resistance. A strict definition of anthracycline resistance implies progressive disease during anthracycline chemotherapy. Among the cytotoxic drugs only 5-Fluorouracil (given as 24 h continuous infusion with folinic acid) and the taxanes produce more than 20% objective remission (RR) in case of anthracycline resistance, whereas the highest response rate was reported for docetaxel (32-57%). Only few randomized studies were performed: docetaxel showed higher anti-tumor activity than methotrexat/5-FU (RR: 42% vs 19%, P

Haemophagocytic lymphohistiocytosis (HLH) is an autosomal recessive disease with histiocytic and lymphocytic infiltrations in multiple organs. Cure seems possible only by allogeneic bone marrow transplantation (BMT), but matched sibling donors (MSD) are restricted and high mortality rates are associated with BMT from unrelated donors (URD). We report on 12 consecutive HLH patients with an improved outcome following URD transplants. Eight patients received BMT from URD, four from MSD. Five patients had signs of active HLH at the time of BMT. The conditioning regimen consisted of 20 mg/kg busulphan, 60 mg/kg VP-16 and 120 mg/kg cyclophosphamide and, in case of URD, 90 mg/kg antithymocyte globulin. The doses of busulphan and VP-16 were reduced during the programme to 16 mg/kg and 30 mg/kg, respectively. Using a fivefold graft-versus-host disease (GVHD) prophylaxis, GVHD was absent or mild in 10, and moderate or severe in two patients undergoing unrelated transplants. One patient with URD experienced graft failure and was retransplanted on day 37. Major toxicities were hepatic veno-occlusive disease in five, capillary leak syndrome in two, pneumonia in three, sepsis in one, severe mucositis in one and seizures in two patients. All patients are alive without HLH after a median follow-up of 24.5 months. One patient has chronic GVHD, another patient has severe retardation. Three patients show slight to moderate development delay. These results indicate that in HLH, BMT from matched unrelated donors should be performed. Incomplete resolution of disease activity need not impede a successful outcome.

We investigated the schedule dependency of G-CSF (10 microg/kg) alone in mobilizing peripheral blood progenitor cells (PBPC) in breast cancer patients. After a median of three cycles (range, 2-6) of anthracycline-based chemotherapy, 49 patients with breast cancer (stage II/III,or = 10+ Ln n = 36; locally advanced/inflammatory n = 8, stage IV (NED) n = 5) underwent PBPC collection after steady-state mobilization either with 1 x 10 microg/kg (n = 27) or with 2 x 5 microg/kg (n = 22) G-CSF daily for 4 consecutive days until completion of apheresis. Apheresis was started on day 5. Priming with 2 x 5 microg/kg resulted in a higher median number of CD34+ cells (5.8 vs 1.9 x 10(6)/kg, P = 0.003), MNC (6.6 vs 2.6 x 10(8)/kg, P0.001) and CFU-GM (6.5 vs 1.3 x 10(4)/kg, P = 0.001) in the first apheresis than with 1 x 10 microg/kg. Also the overall number of collected BFU-E was higher in the 2 x 5 microg group (9.2 vs 3.1 x 10(4)/kg; P = 0.01). After high-dose chemotherapy with cyclophosphamide/thiotepa/mitoxantrone (n = 46) hematopoietic engraftment with leukocyte count1.0/nl was reached in both groups after a median of 10 days (range, 8-15) and with platelets count50/nl after 12 (range, 9-40) and 13 days (range, 12-41), respectively. A threshold of2.5 x 10(6)/kg reinfused CD34+ cells ensured rapid platelet engraftment (12 vs 17 days; P = 0.12). Therefore, the target of collecting2.5 x 10(6) CD34+ cells was achieved in 21/27 (80%) patients of the 1 x 10 microg group and in 21/22 (95%) patients of the 2 x 5 microg/kg group with a median of two aphereses (range, 1-4). None in the 10 microg/kg group, but 6/22 (28%) patients in the 2 x 5 microg/kg group required only one apheresis procedure, resulting in fewer apheresis procedures in the 2 x 5 microg/kg group (mean, 1.8 vs 2.3, P = 0.01). These results demonstrate that priming with 10 microg/kg G-CSF alone is well tolerated and effective in mobilizing sufficient numbers of CD34+ cells in breast cancer patients and provide prompt engraftment after CTM high-dose chemotherapy. G-CSF given 5 microg/kg twice daily (2 x 5 microg) leads to a higher harvest of CD34+ cells and required fewer apheresis procedures than when given 10 microg/kg once daily (1 x 10 microg).

We explored the combination of busulfan/cyclophosphamide/etoposide as conditioning regimen prior to bone marrow transplantation in 31 patients with acute myeloid leukemia (AML) in first complete remission. The preparative regimen consisted of 16 mg/kg busulfan, 30-60 mg/kg VP-16, and 120 mg/kg cyclophosphamide. With a median follow-up of 30.5 months (range, 5-60 months), 25 patients are alive in continuous complete remission. Estimated disease-free survival at 5 years is 80.5%. Death was due to transplant-related toxicity (graft-versus-host disease and cytomegalovirus infection, graft-versus-host disease and pneumonia, sepsis and mucositis, respectively). None of the patients have relapsed. As demonstrated by the results of this analysis, the conditioning regimen busulfan/cyclophosphamide/etoposide is effective and well tolerated in patients with AML in first complete remission. Main nonhematological toxicities were mucositis and hepatotoxicity. The low mortality and relapse rate appears to justify allogeneic bone marrow transplantation for patients with AML in first complete remission who have an HLA-identical donor. Whether this regimen offers a substantial improvement in disease-free and overall survival over presently used regimens warrants further investigation.

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.