ESF-EMBO symposium "molecular biology and innovative therapies in sarcomas of childhood and adolescence" Sept 29–Oct 4, Polonia Castle Pultusk, Poland

Rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) are among the most common pediatric sarcomas (Arndt et al., 2012). Despite sarcomas representing a highly heterogeneous group of tumors, ES and alveolar RMS (ARMS) typically share one common genetic characteristic, namely a specific chromosomal translocation (Helman and Meltzer, 2003; Lessnick and Ladanyi, 2012). These translocations generate fusion proteins, which are composed of two transcription factors (TF). Typically, one TF is a developmentally regulated factor that is essential for proper specification of a given lineage and provides the DNA-binding domain, while the partner TF contributes a transactivation domain that drives aberrant expression of target genes. Based on these common genetic characteristics, the first ESF-EMBO research conference entitled “Molecular Biology and Innovative Therapies in Sarcomas of Childhood and Adolescence” with special focus on RMS and ES was held at the Polonia Castle in Pultusk, Poland. The conference gathered 70 participants from more than 15 countries and several continents representing most research groups that are active in this field

Desmoplastic small round cell tumors : Multimodality treatment and new risk factors

Background To evaluate optimal therapy and potential risk factors. Methods Data of DSRCT patients 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.Peer reviewe

Cytotoxic Capacity of IL-15-Stimulated Cytokine-Induced Killer Cells Against Human Acute Myeloid Leukemia and Rhabdomyosarcoma in Humanized Preclinical Mouse Models

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

Haploidentical hematopoietic stem cell transplantation as individual treatment option in pediatric patients with very high-risk sarcomas

Background Prognosis of children with primary disseminated or metastatic relapsed sarcomas remains dismal despite intensification of conventional therapies including high-dose chemotherapy. Since haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is effective in the treatment of hematological malignancies by mediating a graft versus leukemia effect, we evaluated this approach in pediatric sarcomas as well. Methods Patients with bone Ewing sarcoma or soft tissue sarcoma who received haplo-HSCT as part of clinical trials using CD3+ or TCRα/β+ and CD19+ depletion respectively were evaluated regarding feasibility of treatment and survival. Results We identified 15 patients with primary disseminated disease and 14 with metastatic relapse who were transplanted from a haploidentical donor to improve prognosis. Three-year event-free survival (EFS) was 18,1% and predominantly determined by disease relapse. Survival depended on response to pre-transplant therapy (3y-EFS of patients in complete or very good partial response: 36,4%). However, no patient with metastatic relapse could be rescued. Conclusion Haplo-HSCT for consolidation after conventional therapy seems to be of interest for some, but not for the majority of patients with high-risk pediatric sarcomas. Evaluation of its future use as basis for subsequent humoral or cellular immunotherapies is necessary

Second-line treatment of pediatric patients with relapsed rhabdomyosarcoma adapted to initial risk stratification: Data of the European Soft Tissue Sarcoma Registry (SoTiSaR).

BACKGROUND Outcome of relapsed disease of localized rhabdomyosarcoma remains poor. An individual treatment approach considering the initial systemic treatment and risk group was included in the Cooperative Weichteilsarkom Studiengruppe (CWS) Guidance. METHODS Second-line chemotherapy (sCHT) ACCTTIVE based on anthracyclines (adriamycin, carboplatin, cyclophosphamide, topotecan, vincristine, etoposide) was recommended for patients with initial low- (LR), standard- (SR), and high-risk (HR) group after initial treatment without anthracyclines. TECC (topotecan, etoposide, carboplatin, cyclophosphamide) was recommended after initial anthracycline-based regimen in the very high-risk (VHR) group. Data of patients with relapse (n = 68) registered in the European Soft Tissue Sarcoma Registry SoTiSaR (2009-2018) were retrospectively analyzed. RESULTS Patients of initial LR (n = 2), SR (n = 16), HR (n = 41), and VHR (n = 9) group relapsed. sCHT consisted of ACCTTIVE (n = 36), TECC (n = 12), or other (n = 15). Resection was performed in 40/68 (59%) patients and/or radiotherapy in 47/68 (69%). Initial risk stratification, pattern/time to relapse, and achievement of second complete remission were significant prognostic factors. Microscopically incomplete resection with additional radiotherapy was not inferior to microscopically complete resection (p = .17). The 5-year event-free survival (EFS) and overall survival (OS) were 26% (±12%) and 31% (±14%). The 5-year OS of patients with relapse of SR, HR, and VHR groups was 80% (±21%), 20% (±16%), and 13% (±23%, p = .008), respectively. CONCLUSION Adapted systemic treatment of relapsed disease considering the initial risk group and initial treatment is reasonable. New treatment options are needed for patients of initial HR and VHR groups

Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants.

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors

CD133 Positive Embryonal Rhabdomyosarcoma Stem-Like Cell Population Is Enriched in Rhabdospheres

Cancer stem cells (CSCs) have been identified in a number of solid tumors, but not yet in rhabdomyosarcoma (RMS), the most frequently occurring soft tissue tumor in childhood. Hence, the aim of this study was to identify and characterize a CSC population in RMS using a functional approach. We found that embryonal rhabdomyosarcoma (eRMS) cell lines can form rhabdomyosarcoma spheres (short rhabdospheres) in stem cell medium containing defined growth factors over several passages. Using an orthotopic xenograft model, we demonstrate that a 100 fold less sphere cells result in faster tumor growth compared to the adherent population suggesting that CSCs were enriched in the sphere population. Furthermore, stem cell genes such as oct4, nanog, c-myc, pax3 and sox2 are significantly upregulated in rhabdospheres which can be differentiated into multiple lineages such as adipocytes, myocytes and neuronal cells. Surprisingly, gene expression profiles indicate that rhabdospheres show more similarities with neuronal than with hematopoietic or mesenchymal stem cells. Analysis of these profiles identified the known CSC marker CD133 as one of the genes upregulated in rhabdospheres, both on RNA and protein levels. CD133+ sorted cells were subsequently shown to be more tumorigenic and more resistant to commonly used chemotherapeutics. Using a tissue microarray (TMA) of eRMS patients, we found that high expression of CD133 correlates with poor overall survival. Hence, CD133 could be a prognostic marker for eRMS. These experiments indicate that a CD133+ CSC population can be enriched from eRMS which might help to develop novel targeted therapies against this pediatric tumor