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Purpose: Optimization of local therapies in synovial sarcoma (SS) considered unresectable at diagnosis is needed. We evaluated the effects of neoadjuvant versus adjuvant radiation versus surgery only on long-term outcomes.
Methods: Patients with macroscopic SS tumors before chemotherapy (IRS-group-III) in the trials CWS-81, CWS-86, CWS-91, CWS-96, CWS-2002-P and SoTiSaR-registry were analyzed. Local therapies were scheduled after 3 neoadjuvant chemotherapy cycles.
Results: Median age of 145 patients was 14.5 years. 106 survivors had median follow-up of 7.0 years. Tumor site was 96 extremities, 19 head–neck, 16 shoulder/hip, 14 trunk. Tumors were < 3 cm in 16, 3–5 cm in 28, 5–10 cm in 55, > 10 cm in 34 patients. In a secondary resection during chemotherapy, R0-status was accomplished in 82, R1 in 30, R2 in 21 (12 missing). Radiotherapy was administered to 115 (R0 61, R1 29, R2 20, missing 5), thereof 57 before and 52 after tumor resection. 23 were treated with surgery only. For all patients, 5 year event-free (EFS) and overall survival (OS) was 68.9% ± 7.6 (95%CI) and 79.1% ± 6.9. To establish independent significance, tumor site, size, surgical results and sequencing of local therapies were analyzed in a Cox regression analysis. Variables associated with EFS and OS are site, size and sequencing of local therapies. Variables associated with local recurrence are site, surgical results and sequencing of local therapies. The only variable associated with suffering metastatic recurrence is tumor size.
Conclusion: Differences in sequencing of local therapy procedures are independently associated with outcomes. Best local control is achieved when tumors are irradiated pre-operatively and undergo R0 or R1 resection thereafter.
Background: The benefit of adjuvant therapy in synovial sarcoma (SS) treatment is under debate. Long-term follow-up data are missing.
Methods: SS patients treated in the consecutive trials CWS-81, CWS-86, CWS-91, CWS-96, CWS-2002-P, and the SoTiSaR-registry till 2013 were analyzed.
Results: Median age of 185 patients was 13.9 years (0.1–56)—with median follow-up of 7.4 years for 163 survivors. Most tumors (76%) were located in extremities. Size was < 3 cm in 58 (31%), 3–5 cm in 59 (32%), 5–10 cm in 42 (23%), and > 10 cm in 13 (7%) (13 missing). In 84 (45%) tumors, first excision was complete (R0 corresponding to IRS-I-group) and in 101 (55%) marginal (R1 corresponding to IRS-II-group). In a subsequent surgical intervention during chemotherapy, R0-status was accomplished in 23 additional IRS-II-group patients with secondary surgery. Radiotherapy was administered to 135 (73%), thereof 62 with R0-status and 67 R1-status (6 missing information). Adjuvant chemotherapy was administered to all but six patients. 5-year event-free (EFS) and overall survival (OS) was 82.9% ± 5.7 (95%CI) and 92.5% ± 3.9. Local and metastatic relapse-free survival was 91.3% ± 4.3 and 92.3% ± 4.1 at 5 years, respectively. In the multivariate analysis, tumor size and no chemotherapy were independently associated with EFS. Size and site were associated with OS. In a detailed analysis of local and metastatic events, tumor size was associated with an independent risk for developing metastases. No independent factor for suffering local recurrence could be identified.
Discussion: Omission of chemotherapy in a non-stratified way seems not justified. Size governs survival due to high linear association with risk of suffering metastatic recurrence in a granular classification.
Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug.
Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD.
In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9–14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/−) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(−) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/−)CD69(+)NCR(high)CD62L(−) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.
Pediatric patients with recurrent, refractory or advanced soft tissue sarcoma (STS) who are simultaneously showing signs of cumulative treatment toxicity are in need of novel therapies. In this preclinical analysis, we identified ErbB2 as a targetable antigen on STS cells and used cytokine-induced killer (CIK) cells transduced with the lentiviral 2nd-generation chimeric antigen receptor (CAR) vector pS-5.28.z-IEW to target ErbB2-positive tumors. Solely CIK cell subsets with the CD3+ T cell phenotype showed up to 85% cell surface expression of the respective CAR. A comparison of wildtype (WT), mock-vector and ErbB2-CAR-CIK cells showed, that engineered cells exhibited diminished in vitro expansion, retained WT CIK cell phenotype with higher percentages of differentiated effector memory/effector cells. Activating natural killer (NK) cell receptor NKG2D-restricted target cell recognition and killing of WT and ErbB2-CAR-CIK cells was maintained against ErbB2-negative tumors, while ErbB2-CAR-CIK cells demonstrated significantly increased cytotoxicity against ErbB2-positive targets, including primary tumors. ErbB2-CAR- but not WT CIK cells proliferated, infiltrated and efficiently lysed tumor cell monolayers as well as 3D tumor spheroids.
Here, we demonstrate a potential cell therapeutic approach using ErbB2-CAR-CIK cells for the recognition and elimination of tumor cells expressing ErbB2, which we identified as a targetable antigen on high-risk STS cells.
Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing process (GMP)-compliant protocol for the generation of ready-for-use therapeutic CIK cell doses starting from one unstimulated donor-derived peripheral blood (PB) or leukocytapheresis (LP) products. Culture medium with or without the addition of either AB serum, fresh frozen plasma (FFP) or platelet lysate (PL) was used for culture. Fresh and cryopreserved CIK cells were compared regarding expansion rate, viability, phenotype, and ability to inhibit leukemia growth. Cell numbers increased by a median factor of 10-fold in the presence of FFP, PL, or AB serum, whereas cultivation in FFP/PL-free or AB serum-free medium failed to promote adequate CIK cell proliferation (p < 0.01) needed to provide clinical doses of 1 × 106 T cells/kG, 5 × 106 T cells/kG, 1 × 107 T cells/kG, and 1 × 108 T cells/kG recipient body weight. CIK cells consisting of T cells, T- natural killer (T-NK) cells and a minor fraction of NK cells were not significantly modified by different medium supplements. Moreover, neither cytotoxic potential against leukemic THP-1 cells nor cell activation shown by CD25 expression were significantly influenced. Moreover, overnight and long-term cryopreservation had no significant effect on the composition of CIK cells, their phenotype or cytotoxic potential. A viability of almost 93% (range: 89–96) and 89.3% (range: 84–94) was obtained after freeze-thawing procedure and long-term storage, respectively, whereas viability was 96% (range: 90-97) in fresh CIK cells. Altogether, GMP-complaint CIK cell generation from an unstimulated donor-derived PB or LP products was feasible. Introducing FFP, which is easily accessible, into CIK cell cultures was time- and cost-saving without loss of viability and potency in a 10-12 day batch culture. The feasibility of cryopreservation enabled storage and delivery of sequential highly effective ready-for-use CIK cell doses and therefore reduced the number of manufacturing cycles.
Background: Prolonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context.
Methods: CIK cells with EBV-specificity were generated from peripheral blood mononuclear cells (PBMCs), expanded in the presence of interferon-γ, anti-CD3, interleukin (IL)-2 and IL-15 and were pulsed twice with EBV consensus peptide pool. CIK cells with EBV-specificity and conventional CIK cells were phenotypically and functionally analyzed. Additionally, CIK cells with EBV-specificity were applied to a patient with EBV-related PTLD rapidly progressing to highly aggressive B-cell lymphoma on a compassionate use basis after approval and agreement by the regulatory authorities.
Results: Pre-clinical analysis showed that generation of CIK cells with EBV-specificity was feasible. In vitro cytotoxicity analyses showed increased lysis of EBV-positive target cells, enhanced proliferative capacity and increased secretion of cytolytic and proinflammatory cytokines in the presence of EBV peptide-displaying target cells. In addition, 1 week after infusion of CIK cells with EBV-specificity, the patient's highly aggressive B-cell lymphoma persistently disappeared. CIK cells with EBV-specificity remained detectable for up to 32 days after infusion and infusion did not result in acute toxicity.
Discussion: The transfer of both anti-cancer potential and T-cell memory against EBV infection provided by EBV peptide-induced CIK cells might be considered a therapy for EBV-related PTLD.
Prognosis of refractory childhood cancers despite multimodal treatment strategies remains poor. Here, we report a single center experience encountered in 18 patients with refractory solid malignancies treated with adoptive cellular immunotherapy (ACI) from haploidentical or matched donors following hematopoietic stem cell transplantation. While seven patients were in partial and six in complete remission (CR), five patients suffered from relapsed diseases at the time of ACI. 1.5-year probabilities of overall survival (OS) and progression-free survival (PFS) were 19.5% and 16.1% for all patients. Patients in CR showed estimated 1.5-year OS and PFS of 50.1% and 42.7%, respectively. CR was induced or rather sustained in ten children, with two still being alive 9.6 and 9.3 years after ACI. Naïve, central and effector memory T-cells correlated with responses. However, the majority of patients relapsed. Cumulative incidence of relapse was 79.8% at 1.5 years. Acute graft versus host disease (aGVHD) occurred in nine of 18 patients (50%) with aGVHD grade I–II observed in six (33%) and aGVHD grade III seen in three (17%) patients, manageable in all cases.
Altogether, study results indicate that donor-derived ACI at its current state offers palliation but no clear curative benefit for refractory childhood cancers and warrants further improvement.
Background: Cytokine-induced-killer (CIK) cells are a promising immunotherapeutic approach for impending relapse following hematopoietic stem cell transplantation (HSCT). However, there is a high risk for treatment failure associated with severe graft versus host disease (GvHD) necessitating pharmaceutical intervention post-transplant. Whether immunosuppression with mycophenolate mofetil (MMF) or Ciclosporin A (CsA) influences the cytotoxic effect of CIK cell immunotherapy is still an open issue.
Methods: CIK cells were generated from PBMC as previously described followed by co-incubation with mycophenolic acid (MPA) or CsA. Proliferation, cytotoxicity and receptor expression were investigated following short- (24 h), intermediate- (3 days) and long-term (7 days) MPA incubation with the intention to simulate the in vivo situation when CIK cells were given to a patient with relevant MPA/CsA plasma levels.
Results: Short-term MPA treatment led to unchanged proliferation capacity and barely had any effect on viability and cytotoxic capability in vitro. The composition of CIK cells with respect to T-, NK-like T- and NK cells remained stable. Intermediate MPA treatment lacked effects on NKG2D, FasL and TRAIL receptor expression, while an influence on proliferation and viability was detectable. Furthermore, long-term treatment significantly impaired proliferation, restricted viability and drastically reduced migration-relevant receptors accompanied by an alteration in the CD4/CD8 ratio. CD3+CD56+ cells upregulated receptors relevant for CIK cell killing and migration, whereas T cells showed the most interference through significant reductions in receptor expression. Interestingly, CsA treatment had no significant influence on CIK cell viability and the cytotoxic potential against K562.
Conclusions: Our data indicate that if immunosuppressant therapy is indispensable, efficacy of CIK cells is maintained at least short-term, although more frequent dosing might be necessary.