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Background: Biliary rhabdomyosarcoma (RMS) is the most common biliary tumor in children. The management of affected patients contains unique challenges because of the rarity of this tumor entity and its critical location at the porta hepatis, which can make achievement of a radical resection very difficult.
Methods: In a retrospective chart analysis we analysed children suffering from biliary RMS who were registered in three different CWS trials (CWS-96, CWS-2002P, and SoTiSaR registry).
Results: Seventeen patients (12 female, 5 male) with a median age of 4.3 years were assessed. The median follow-up was 42.2 months (10.7–202.5). The 5-year overall (OS) and event free survival (EFS) rates were 58% (45–71) and 47% (34–50), respectively. Patients > 10 years of age and those with alveolar histology had the worst prognosis (OS 0%). Patients with botryoid histology had an excellent survival (OS 100%) compared to those with non-botryoid histology (OS 38%, 22–54, p = 0.047). Microscopic complete tumor resection was achieved in almost all patients who received initial tumor biopsy followed by chemotherapy and delayed surgery.
Conclusion: Positive predictive factors for survival of children with biliary RMS are age ≤ 10 years and botryoid tumor histology. Primary surgery with intention of tumor resection should be avoided.
Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite therapeutic progress, prognosis in high-risk NB is poor and innovative therapies are urgently needed. Therefore, we addressed the potential cytotoxic capacity of interleukin (IL)-activated natural killer (NK) cells compared to cytokine-induced killer (CIK) cells for the treatment of NB. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by indirect CD56-enrichment or CD3/CD19-depletion and expanded with different cytokine combinations, such as IL-2, IL-15, and/or IL-21 under feeder-cell free conditions. CIK cells were generated from PBMCs by ex vivo stimulation with interferon-γ, IL-2, OKT-3, and IL-15. Comparative analysis of expansion rate, purity, phenotype and cytotoxicity was performed. CD56-enriched NK cells showed a median expansion rate of 4.3-fold with up to 99% NK cell content. The cell product after CD3/CD19-depletion consisted of a median 43.5% NK cells that expanded significantly faster reaching also 99% of NK cell purity. After 10–12 days of expansion, both NK cell preparations showed a significantly higher median cytotoxic capacity against NB cells relative to CIK cells. Remarkably, these NK cells were also capable of efficiently killing NB spheroidal 3D culture in long-term cytotoxicity assays. Further optimization using a novel NK cell culture medium and a prolonged culturing procedure after CD3/CD19-depletion for up to 15 days enhanced the expansion rate up to 24.4-fold by maintaining the cytotoxic potential. Addition of an IL-21 boost prior to harvesting significantly increased the cytotoxicity. The final cell product consisted for the major part of CD16−, NCR-expressing, poly-functional NK cells with regard to cytokine production, CD107a degranulation and antitumor capacity. In summary, our study revealed that NK cells have a significantly higher cytotoxic potential to combat NB than CIK cell products, especially following the synergistic use of IL-15 and IL-21 for NK cell activation. Therefore, the use of IL-15+IL-21 expanded NK cells generated from CD3/CD19-depleted apheresis products seems to be highly promising as an immunotherapy in combination with haploidentical stem cell transplantation (SCT) for high-risk NB patients.
In the current study we compared the molecular signature of expanded mesenchymal stromal cells (MSCs) derived from selected CD271+ bone marrow mononuclear cells (CD271-MSCs) and MSCs derived from non-selected bone marrow mononuclear cells by plastic adherence (PA-MSCs). Transcriptome analysis demonstrated for the first time the upregulation of 115 and downregulation of 131 genes in CD271-MSCs. Functional enrichment analysis showed that the upregulated genes in CD271-MSCs are significantly enriched for extracellular matrix (tenascin XB, elastin, ABI family, member 3 (NESH) binding protein, carboxypeptidase Z, laminin alpha 2 and nephroblastoma overexpressed) and cell adhesion (CXCR7, GPNMB, MYBPH, SVEP1, ARHGAP6, TSPEAR, PIK3CG, ABL2 and NCAM1). CD271-MSCs expressed higher gene transcript levels that are involved in early osteogenesis/chondrogenesis/adipogenesis (ZNF145, FKBP5). In addition, increased transcript levels for early and late osteogenesis (DPT, OMD, ID4, CRYAB, SORT1), adipogenesis (CTNNB1, ZEB, LPL, FABP4, PDK4, ACDC), and chondrogenesis (CCN3/NOV, CCN4/WISP1, CCN5/WISP2 and ADAMTS-5) were detected. Interestingly, CD271-MSCs expressed increased levels of hematopoiesis associated genes (CXCL12, FLT3L, IL-3, TPO, KITL). Down-regulated genes in CD271-MSCs were associated with WNT and TGF-beta signaling, and cytokine/chemokine signaling pathways. In addition to their capacity to support hematopoiesis, these results suggest that CD271-MSCs may contain more osteo/chondro progenitors and/or feature a greater differentiation potential.
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: To evaluate optimal therapy and potential risk factors.
Methods: Data of DSRCT patients <40 years treated in prospective CWS trials 1997‐2015 were analyzed.
Results: Median age of 60 patients was 14.5 years. Male:female ratio was 4:1. Tumors were abdominal/retroperitoneal in 56/60 (93%). 6/60 (10%) presented with a localized mass, 16/60 (27%) regionally disseminated nodes, and 38/60 (63%) with extraperitoneal metastases. At diagnosis, 23/60 (38%) patients had effusions, 4/60 (7%) a thrombosis, and 37/54 (69%) elevated CRP. 40/60 (67%) patients underwent tumor resection, 21/60 (35%) macroscopically complete. 37/60 (62%) received chemotherapy according to CEVAIE (ifosfamide, vincristine, actinomycin D, carboplatin, epirubicin, etoposide), 15/60 (25%) VAIA (ifosfamide, vincristine, adriamycin, actinomycin D) and, 5/60 (8%) P6 (cyclophosphamide, doxorubicin, vincristine, ifosfamide, etoposide). Nine received high‐dose chemotherapy, 6 received regional hyperthermia, and 20 received radiotherapy. Among 25 patients achieving complete remission, 18 (72%) received metronomic therapies. Three‐year event‐free (EFS) and overall survival (OS) were 11% (±8 confidence interval [CI] 95%) and 30% (±12 CI 95%), respectively, for all patients and 26.7% (±18.0 CI 95%) and 56.9% (±20.4 CI 95%) for 25 patients achieving remission. Extra‐abdominal site, localized disease, no effusion or ascites only, absence of thrombosis, normal CRP, complete tumor resection, and chemotherapy with VAIA correlated with EFS in univariate analysis. In multivariate analysis, significant factors were no thrombosis and chemotherapy with VAIA. In patients achieving complete remission, metronomic therapy with cyclophosphamide/vinblastine correlated with prolonged time to relapse.
Conclusion: Pleural effusions, venous thrombosis, and CRP elevation were identified as potential risk factors. The VAIA scheme showed best outcome. Maintenance therapy should be investigated further.
(1) Background: Refractory acute graft-versus-host disease (R-aGvHD) remains a leading cause of death after allogeneic stem cell transplantation. Survival rates of 15% after four years are currently achieved; deaths are only in part due to aGvHD itself, but mostly due to adverse effects of R-aGvHD treatment with immunosuppressive agents as these predispose patients to opportunistic infections and loss of graft-versus-leukemia surveillance resulting in relapse. Mesenchymal stromal cells (MSC) from different tissues and those generated by various protocols have been proposed as a remedy for R-aGvHD but the enthusiasm raised by initial reports has not been ubiquitously reproduced.
(2) Methods: We previously reported on a unique MSC product, which was generated from pooled bone marrow mononuclear cells of multiple third-party donors. The products showed dose-to-dose equipotency and greater immunosuppressive capacity than individually expanded MSCs from the same donors. This product, MSC-FFM, has entered clinical routine in Germany where it is licensed with a national hospital exemption authorization. We previously reported satisfying initial clinical outcomes, which we are now updating. The data were collected in our post-approval pharmacovigilance program, i.e., this is not a clinical study and the data is high-level and non-monitored.
(3) Results: Follow-up for 92 recipients of MSC-FFM was reported, 88 with GvHD ≥°III, one-third only steroid-refractory and two-thirds therapy resistant (refractory to steroids plus ≥2 additional lines of treatment). A median of three doses of MSC-FFM was administered without apparent toxicity. Overall response rates were 82% and 81% at the first and last evaluation, respectively. At six months, the estimated overall survival was 64%, while the cumulative incidence of death from underlying disease was 3%.
(4) Conclusions: MSC-FFM promises to be a safe and efficient treatment for severe R-aGvHD.
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.