The authors report on 2 boys, 11(1/2) and 13 years old, who received allogeneic stem cell transplantation (alloSCT) from their HLA-identical sibling after relapse of stage IV alveolar rhabdomyosarcoma. Both patients were transplanted in a non-remission status. After alloSCT both patients experienced disease progression at the primary tumor location sites and died due to the underlying disease 146 and 379 days after transplantation. The authors conclude that an alloSCT derived graft versus tumor effect might not be effective enough to overcome alveolar rhabdomyosarcoma when transplantation is carried out in a nonremission status.

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In multiple myeloma (MM) patients allogeneic blood stem cell transplantation (allo- BSCT) from unrelated donors can induce prolonged disease control through induction of a graft-versus-myeloma effect. The use of reduced intensity conditioning (RIC) regimens prior to allogeneic transplantation reduces the high treatment related mortality although such approaches are limited by morbidity and mortality due to acute and chronic GvHD. 8 patients with MM, 5 males and 3 females, with a median age of 63 years (range 51 to 71) underwent allogenic transplantation because of aggressive or refractory disease after induction therapy. Peripheral blood stem cells from a matched unrelated donor were used in all patients after reduced intensity conditioning (RIC) with either treosulfan/fludarabin (n=1) or 2Gy/fludarabin (n=2) or fludarabin/melphalan (n=5). All patients received ATG within conditioning therapy. Five patients had a fully matched donor. Two donors had a minor-subtype mismatch and one patient was grafted from a donor with a major mismatch. Median follow up of surviving patients is 24 months (range 3–28). Acute GvHD grade III/IV was not seen in any patient whereas the incidence of acute GvHD grade I/II was 50%. Day +100 mortality was 38% when one patient died because of progressive disease 7 months after transplantation, one patient died because of EBV-related lymphoproliferative disease 2 months after transplantation and one patient died because of cerebral CMV infection 3 months after transplantation. 5 out of 8 patients transplanted are alive, 4 in complete clinical remission and 1 patient developed progressive disease 13 months after transplantation. Here we report our data on the molecular monitoring of minimal residual disease in these patients. Residual lymphoma cells were detected by quantitative real-time PCR in blood and bone marrow samples before and after transplant clone-specific with VDJ-CDR-3 rearrangements serving as molecular targets. All pre-therapeutic bone marrow and peripheral blood samples tested contained 0.1 – 90 % myeloma cells. It is notable that bone marrow samples contained in average 10 times more myeloma cells as detected by quantitative PCR. In general, a 0.5 – 3 log reduction of myeloma cells was achieved within the first 2–3 months after transplantation within the bone marrow whereas a 2 – 4 log reduction of circulating myeloma cells was achieved. Both patients being in complete clinical remission 24 respectively 28 months after transplantation had achieved a continuing complete molecular remission. In these two patients the residual myeloma cells were detected within the first 6 months after transplantation. The immunosuppressive therapy was adapted and reduced in both cases with the consequence that the residual myeloma cells disappeared and could not further be detected at a sensitivity of 1/105–6. The high amount of myeloma cells in the bone marrow in one patient was only reduced by about 0.5 log after transplantation. Three additional donor lymphocyte (DLI) infusions were administered 3 – 6 months after transplantation. The patient developed grade 2 acute GvHD after DLI. As a consequence the residual amount of myeloma cells was reduced by 4 log but was still detectable. Prior to the clinical extramedullary relapse of multiple myeloma an increasing amount of myeloma cells was detected within the bone marrow (2 months before) and within the peripheral blood (3 months before). In a further patient residual myeloma cells (0.02%) could be detected about 4 weeks after transplantation but rapidly increased to 20% within further 4 weeks when clinical relapse became evident. Myeloma was controlled by treatment with bortezomib and thalidomide for a period of 5 months after which patient died because of progressive disease. These results strongly favour the idea of potent graft versus myeloma effects in patients with MM given an allogeneic transplant. In conclusion, allogeneic blood stem cell transplantation after RIC seems to be an attractive treatment for patients with MM which can induce myeloma eradication and prolonged disease control as measured by complete clinical as well as molecular remissions.

In a prospective multicenter phase III trial (M 39023) advanced stage follicular lymphoma (FL) patients were randomized to receive eight cycles of MCP (mitoxantrone, chlorambucil, prednisolone) chemotherapy alone or in combination with rituximab (R-MCP). This study was carried out to determine the effects of this therapy on circulating lymphoma cells (CLC) and to assess the value of CLC quantitation as a molecular marker of disease activity and as a prognostic parameter. CLC numbers were determined by real-time quantitative PCR for the t(14;18)-MBR translocation or by allele-specific PCR for rearranged immunoglobulin heavy chain genes. Quantitative PCR of a reference gene (wild type K-ras) allowed the exact quantitation of cells tested per sample and to exclude samples with insufficient DNA content (< 500,000 cells). We analyzed serial blood samples from 43 patients obtained before, during and after completion of therapy. Baseline clinical characteristics and response to therapy of the 43 patients of this study were not significantly different to all 201 FL patients of the clinical trial except for a higher complete response (CR) rate in the R-MCP group (18/25 (72%) in this study versus 52/105 (50%) in the clinical trial, p=.04). Similar to the results of the clinical study, response rate and CR rate in the present study were significantly higher in the R-MCP arm than after therapy with MCP alone (25/25 (100%) versus 13/18 (72%), p= .009, and 18/25 (72%) versus 5/18 (28%), p= .006, respectively). Clearance of CLC at the end of therapy was achieved in 21/25 patients (84%) treated with R-MCP compared to 0/18 (0%) after MCP alone (p< .0001). R-MCP patients achieved a greater reduction of CLC after completion of therapy and a greater reduction of CLC per treatment cycle than patients treated with MCP alone, even if the comparison was restricted to patients with clinical response (3.88 log and 1.18 log reduction versus 2.21 log and 0.23 log reduction, p= .001 and p< .0001). A ≥ 2 log reduction of CLC after completion of therapy was associated with a favourable clinical response (p= 0.0007) and prolonged event-free survival (p= 0.02) regardless of treatment arm. Among patients with a ≥2 log CLC reduction there was no significant difference in EFS between patients with PCR negative samples and patients with PCR positive blood samples at completion of therapy (p= 0.091). In conclusion, R-MCP led to a rapid and sustained eradication of CLC in the majority of patients. The results of serial determinations of CLC numbers showed a good correlation with the quality and duration of the clinical response. Therefore, quantitative molecular disease monitoring could help to develop individualized treatment strategies for patients with advanced stage FL.

Immunization of patients with B-cell non-Hodgkin’s lymphoma with the individual idiotype expressed by the malignant clone can induce tumor-specific immune responses that correlate with improved remission duration and survival rates after cytoreductive chemotherapy. We have developed a production method for a recombinant idiotype vaccine based on expression of Fab fragments in E. coli. Intradermal administration of this vaccine mixed with lipid-based adjuvant and subcutaneous coadministration of GM-CSF has excellent immunogenicity as demonstrated by a high and idiotype-specific immune response rate in a phase I trial in advanced lymphoma patients with a severely impaired immune status (Bertinetti et al., Cancer Research 2006). Of a cohort of 15 patients with newly diagnosed or relapsed indolent B cell malignancies who had not been treated with conventional cytoreductive therapy and received 6 monthly immunizations of this idiotype vaccine, we observed 6 cases (40%) with objective quantitative evidence of tumor regression. 5 of these patients were in primary “watch and wait” situation for indolent NHL, and 1 follicular lymphoma patient had recurrence of nodal disease after a complete remission achieved in the phase I trial. At weeks 0, 8, 24, 36, and 52 after initiation of vaccination, staging examinations were performed including enumeration of circulating tumor cells by quantitative real-time PCR, and immune responses were assessed by ELISPOT and ELISA. Beginning with the 4th vaccination, evidence for reduction of nodal disase was noted in 5 cases, 4 of which achieved an objective partial response after all 6 scheduled immunizations according to standard staging criteria. These patients received continuing vaccinations in intervals of 1–3 months, and no progression has occurred after a follow-up of 9–32 months. Of 6 patients who had detectable lymphoma cells in the peripheral blood, 4 showed a continuous, at least 10fold decrease of the number of circulating lymphoma cells during the course of the vaccination. Two of the partial responders had never evidence for circulating tumor cells. A T cell response to the vaccine developed in 4/5 evaluated patients with evidence of tumor regression and in 3/6 patients without evidence for a clinical response. These cellular responses were maintained at stable levels during the vaccination and continued to be present 6 months after the last vaccination. Humoral immune responses were induced in 5/5 patients with declining tumor burden and in 2/6 non-responders. In 3 cases, the anti-idiotypic antibody titers declined after 6 months. In cases where anti-idiotype antibodies of IgM and IgG isotype occurred, IgM preceded the appearance of IgG. All objective clinical responders mounted a combined cellular and humoral immune response. The temporal association of immune responses with tumor regression provides support for the assumption that recombinant idiotype vaccination in the chosen format may play a causal role for a favorable disease course in previously untreated indolent lymphomas and may hence obviate the need for cytoreductive chemotherapy for extended time periods.