Genetic variation is one of the main characteristics of pediatric sarcomas. This is mostly explained because despite being originated from a mesenchymal cell, they constitute different histologic entities with different genomic landscapes that explain their unequal behaviours. Beside pathology, chromosomal segmental aberrations,16 changes in ploidy and specific gene alterations are routinely used in order to guide intensity of treatment in pediatric oncology protocols.
It is worth noting important differences spotted when comparing adult with pediatric NGS studies in sarcomas.17 Epidemiologically, sarcomas represent less than 1% of all solid malignant cancers in adult population while they represent 20% of all pediatric solid malignant cancers. Therefore, the first main difference lies in the fact that the magnitude of the problem is proportionally much higher in pediatric population. Furthermore, adult type cancers such as epithelial neoplasms arise after accumulation of multiple sequential mutations directly linked to environmental exposures, and arise within differentiated adult tissues18,19. Mesenchymal tumors, such as sarcomas appear both in adult and pediatric population. However, specific histologic subtypes and clinical progression are age-dependent, suggesting differential pathogenetics and underlying molecular mechanisms for tumor initiation and clinical behavior in the different age subgroups18.
In this study, we found that the overall mutational load in our cohort was relatively low when compared to adult studies. This might be explained by the fact that adult sarcomas are mostly driven by mutagenic exposure from environmental factors, whereas most of pediatric cancers contain a relatively small number of mutations20 and frequently display unique gene rearrangements. Although this restricts the targeted treatment to available drugs, it also makes them attractive candidates for drug discovery15.
In order to improve outcome, international efforts amongst cooperative groups have been carried out developing genomic precision medicine programs. These programs aim to bring NGS approaches into the clinical practice and require the identification of patients that might benefit from targeted therapies. Once these targets are identified, in pediatric population it is important to communicate these results, as well as possible toxicities observed by compassionate use basis as dosing is more complex when compared to adult population. Hence, the importance of promoting pediatric phase I clinical trials in order to titrate infant dosing.
In this study, we conclude that the most frequent somatic mutation observed in pediatric sarcomas occurs in TP53 (27% of the pathogenic mutations detected by NGS). This information correlates with adult sarcoma cohorts such as the study presented by Groisberg et al.21 Xiaosheng et al22 compared overall survival (OS) time between TP53-mutated and TP53-wildtype cancers in 20 adult cancer types. They reported that patients with TP53 mutations had lower survival compared with those without TP53 mutations in colon, lung and pancreas adenocarcinoma, acute myeloid leukemia and other epithelial cancers. In pediatric oncology, the clinical significance of somatic TP53 mutations remains unrecognized and no routine testing or therapy intensification is considered. Recent studies suggest that mutation in TP53 in localized EWS is not a reliable prognostic marker23. In order to target TP53, small molecules that reactivate mutant p53 by restoring wild-type conformation have been identified by various approaches. APR-246 alone is currently being tested in prostate or ovarian cancers or in combination with azacitidine in myeloid malignancies in adult phase I-II trials. No studies are currently recruiting pediatric population.
Mutations in Fibroblast Growth Factor Receptor 4 (FGFR4) have also been described in pediatric sarcomas, most outstandingly in RMS. Higher FGFR4 expression in RMS has been associated with advanced-stage cancer and poor survival24. FGFR4 pathogenic mutations appear in 33% of the embryonal RMS studied in our cohort and all of them received a targeted recommendation therapy. FGFR4 codifies for a cell surface tyrosine kinase (TK) receptor that is involved in normal myogenesis and muscle regeneration. It has been reported that human embryonal RMS cells have increased FGFR4 mRNA expression compared to normal human myoblasts, and FGFR4 pathway blockade decreases proliferation.25 In fact, over-expression and mutational activation of FGFR4 has been reported in RMS, promoting tumor progression. FGFR4 signaling is also a common mechanism of oncogenesis in fusion positive RMS (usually alveolar subtype)25.
Alterations in FGFR4 are clinically relevant because they are actionable targets in patients with RMS. New generation of multikinase inhibitors are under current development such as ponatinib (AP-24534), an orally administered TK inhibitor that was initially developed as an inhibitor for BCL-ABL. Ponatinib recently received FDA approval for the treatment of adult patients with Philadelphia chromosome positive acute lymphoblastic leukemia and chronic myeloid leukemia resistant to other TK inhibitors. Inhibition profile of ponatinib includes other TK such as c-KIT, PDGFR, FLT3, SRC and FGFR26. Moreover, inhibition of FGFR family members with ponatinib has been demonstrated in preclinical models with bladder cancer, endometrial cancer, breast, lung and colon cancer. Samuel Q. Li et al26 tested a panel of RMS cell lines over-expressing FGFR4, all of them exhibiting sensitivity to five different TK inhibitors including ponatinib, cediranib, nintedanib, dovitinib and danusertib. They observed that ponatinib resulted to be the most powerful FGFR4 inhibitor, inhibiting both, mutated and wild-type FGFR4 cell growth. It also inhibited tumor development expressing FGFR4 in vivo26. Currently, ponatinib is being tested in clinical trials including pediatric patients (NCT03934372)25,27. Erdafitinib is also being tested in a phase II trial for tumors with FGFR mutations. (NCT03210714).
The CTNNB1 gene provides instructions to form the protein beta-catenin. The relationship between the Wnt/beta-catetin signaling pathway and desmoid-type fibromatosis (DTF) has been widely studied and it has been reported that the vast majority of DTF tumors (up to 85%) harbor a mutation in exon 3 of the CTNNB1 gene (beta-catetenin)28. These mutations lead to an abnormally stable beta-catenin protein that is more resistant to proteolytic degradation and accumulates within the cells. Excess of beta-catenin promotes an uncontrolled proliferation of cells, allowing the formation of DTF29.
Therapeutic options targeting Wnt/betacatenin signaling pathway are limited and have not been tested in pediatric population. Accumulation of beta-catenin in the nucleus triggers transcription of Wnt-specific genes responsible for the control of cell fate decisions. The development of drugs targeting mutated or altered beta-catenin signaling, or its interaction with CBP, TCF, GSK3β or APC (which are essential to complete its function) has been difficult due to the toxicity of the new compounds. Several of them are currently in Phase 1 clinical trials, such as the PRI-724 molecule (NCT01302405, NCT02413853, NCT01764477, and NCT01606579) that prevents the interaction of beta-catenin with CBP. Despite these and other approaches, there are no clinical trials available for pediatric patients with Wnt/beta-catenin inhibitors.30 All DTF studied in our cohort harbored mutations in CTNNB1.
In the study, a patient with malignant nerve sheath tumor and ATM mutation was treated with PARP inhibitors in combination with olaparib. The ataxia telangiectasia gene (ATM), localized in 11q22-q23, plays an important role in maintaining genomic integrity. It regulates the double-strand DNA breaks repair and activates different checkpoints in the cell cycle. ATM is associated with some types of leukemia and lymphoma and it has also been described in neuroblastoma with 11q deletion. Poly ADP-ribose polymerase (PARP) is a protein that signals DNA damage and contributes towards DNA repair31. PARP catalyzes the addition of ADP-ribose to DNA, helicases, topoisomerases and histones. It also has a critical role in transcription, cellular replication, gene regulation, differentiation, spindle maintenance and protein degradation. PARP inhibition produces persistent single strand DNA breaks leading to double strand DNA breaks and finally produces DNA damage leading to apoptosis and cell cycle arrest. Preclinical studies show that ATM mutated neuroblastoma cells also succumb to apoptosis when treated with PARP inhibitors and neuroblastomas with 11q deletion are extremely sensitive to conventional chemotherapy combined with PARP inhibitors. The patient in the study managed a short period of stable disease but progressed rapidly afterwards31. Other mutations considered as uncertainly significant in ATM have been detected but no recommendations were issued because no previous clinical evidence was found. Currently, early phase trials with PARP inhibitors are recruiting pediatric patients with diverse malignancies.
Recent studies in RMS have revealed recurrent mutations in the RAS pathway, particularly affecting NRAS. Dolghik et al32 demonstrated that PIK3CA played a critical role in the activation of the PI3K/AKT/mTOR pathway in NRAS mutant RMS. They noted that NRAS-mutated RMS cells particularly relied on PIK3CA to prevent cell death upon NRAS silencing or MEK inhibition. Their data showed that specific PIK3CA knockdown was sufficient to cooperatively trigger cell death together with pharmacological MEK inhibition. In addition, pharmacological inhibitors of MEK or NRAS knockdown synergize with the PIK3CA specific inhibitor BYL719 to trigger cell death in NRAS-mutated RMS cells. All this data supports the rationale for the combination of MEK and PIK3CA specific inhibitors in NRAS mutated RMS. This recommendation is a future option for one of the patients studied in our cohort.
In this study, a patient diagnosed with c-KIT positive (CD-117) GIST was treated with imatinib and so far, has maintained complete response after surgery. Another patient with ALK + myofibroblastic inflammatory tumor received treatment with ceritinib obtaining a partial response. Both of these rare sarcomas have a classical alteration that has been widely reported before.
In conclusion, we have observed that the incorporation of NGS results together with ancillary studies into pediatric sarcoma clinical practice is feasible and allows personalized treatments with acceptable disease control rates in the relapse setting. At the moment, as the integration NGS as a routine diagnostic technique has been limited this is difficult to estimate, although the situation is changing and sequencing studies are gradually becoming widespread33,34,35. Further investigations are required to confirm this hypothesis.
In this study, up to 23% of the patients would obtain clinical benefit by implementing this precision medicine approach complementing routine diagnostic techniques. Although the understanding of pediatric sarcomas’ biology has improved in a relatively short period of time, outcomes in high-risk tumors remain poor and regarding new therapeutic strategies, very few advances have been highlighted. This emphasizes that strong, international efforts are still required in order to improve implementation of new diagnostic techniques, impulse pediatric drug development and access to clinical trials in childhood. Finally, we would like to stress the importance of treating childhood, adolescent and young adult sarcomas and other types of cancers in specialized units, with all the available expertise and distinct requirements involving this particular population.