Pathophysiology of treatment refractoriness is an evolving area of research and there are a lot of knowledge gaps. The studies have focussed on cancer stem cells (CSC), signalling pathways and multidrug resistance. OGS treatment refractoriness has been relatively well studied compared to others.

Gibbs et al16 demonstrated CSC in OGS which has the ability to self-renew, form sarcospheres and were multipotent. These cells had high expression of CD 133, CD 117 and Stro-1.17,18 In vitro studies in OGS cell lines and xenograft mouse model has shown that exposure to chemotherapy transforms a subpopulation of OGS cells to CSC like phenotype.19 These transformed cells have increased the capacity to form sarcospheres in vitro and initiation of tumour in vivo.20 These transformed cells are chemoresistant and are found in refractory OGS.21

It is thought that Wnt-β-catenin signalling pathway plays a key role in the development of OGS and chemotherapy resistance. Increased cytoplasmic expression β-catenin predicts lung metastasis in murine OGS cell line.22 Small interfering RNA (siRNA) mediated silencing of β-catenin leads to chemoresistance to doxorubicin mediated by N-kappaB, inhibition of invasion and motility in human OGS cell line.23 Increased expression of TWIST decreases β-catenin via PI3K pathway leading to cisplatin sensitivity in vitro.24 Knocking down of β-catenin results in methotrexate sensitivity in OGS cell line.25 Apart from Wnt-β-catenin pathway, Hedgehog pathway, Notch, MAP kinase and FGF signalling pathway play a role in maintaining OGS CSC.26

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Multidrug resistance in OGS is mediated through different mechanisms. ATP-binding cassette transporters play a key role in drug uptake and transport. Intracellular concentration of the drug is decreased by P-glycoprotein (P-gp), a membrane efflux pump.

P-gp is associated with multidrug resistance in OGS cell lines.27 High P-gp levels have been associated with poor event-free survival and increase the risk of adverse events.28 P-gp has a potential to be used as a biomarker for risk stratification in OGS. Polymorphisms in multidrug resistance-associated protein 2 have been associated with necrosis, methotrexate resistance, myelosuppression and cardiotoxicity.29

Methotrexate is one of the key drugs in OGS and it enters into cells through reduced folate carrier (RFC). Reduced RFC expression is associated with poor histologic response.30,31 Low levels of DNA topoisomerase II β are associated with multidrug resistance in OGS cell line.31 Human glutathione S-transferase P1 (GSTP1) overexpression is associated with poor histological response and prognosis, as it metabolizes chemotherapeutic agents.32,33 There is evidence to suggest that OGS cells develop resistance due to the ability to repair the DNA damaged by cisplatin. Excision repair cross-complementing (ERCC) polymorphisms were associated with event-free survival (EFS) in OGS patients.34–36 In a meta-analysis of 858 OGS patients, ERCC2 polymorphism Lys751Gln was associated with OS and His46His mutation was associated with EFS.36 A key enzyme in the base excision repair pathway (repairs DNA damage) is Apurinic/apyrimidinic exonuclease 1 (APEX 1). Increased expression of APEX 1 is associated with decreased disease-free survival and increased tumour recurrence.37 MicroRNAs modulate OGS drug resistance via DNA damage repair, apoptosis avoidance, suppression of autophagy, activation of cancer stem cells and alteration of OGS associated signal pathways.38

EWS therapy resistance is explained on the basis of CSC, drug-metabolizing enzymes and modulation of signalling pathways. It is thought that EWSCSC arises from primitive mesenchymal stem cells.39 The proposed explanations for chemoresistance of EWS CSC are that they are quiescent, have enhanced ability to repair DNA damage, disrupted apoptotic pathways and high expression of drug-efflux proteins.40 Glutathione S transferases (GST) which belong to the family of Phase II detoxification enzymes are involved in the metabolism of toxic compounds. Low expression of microsomal GST was associated with better prognosis and is correlated with sensitivity to doxorubicin. The same group identified molecular signatures using microarray technology which predicted tumour resistance.41 ERBB4 tyrosine kinase is overexpressed in EWS cell line of chemoresistant and metastatic patients. Overexpression of ERBB4 correlates with poor disease-free survival.42 STAG2 overexpression is associated with metastaticEWS.43 Tumour heterogeneity in tumour cells and microenvironment has also been implicated in chemoresistant EWS.40

Constitutional activation of STAT3 causes resistance to chemotherapy in RMS cell lines.44 In a study that included 31 patients of RMS and 12 patients of NRSTS, biopsy was done at baseline from the primary lesion and from the residual tumour at the end of treatment. The paired samples were analysed for P-gp, multidrug resistance-associated protein 1 (MRP 1) and multidrug resistance 3 (MDR 3). Expression of MRP1, MDR 3 and P-gp was higher than in post-treatment specimens suggesting their role in chemoresistance. Clonal selection of MDR protein-expressing tumour cells and up-regulation of MDR proteins were thought to be the possible explanations for increased expression of MDR proteins in post-treatment specimens.45 Serum and glucocorticoid inducible kinase expression in RMS has been shown to be associated with treatment resistance in RMS cell lines.46 In vitro and in vivo studies in RMS have shown that GST mediates chemotherapy resistance.47


Whole genome sequencing was done in 100 patients of OGS with outcome of relapse, percent tumour necrosis and survival. Intronic and intergenic hotspot regions from 26 genes were identified which were associated with relapse. Mutations in genes belonging to AKR enzyme family, PI3K pathways, cell-cell adhesion processes and variants of SLC22 were associated with tumour necrosis and survival.48 In whole-exome analysis of eight OGS patients, out of which three were nonresponder fifteen genes were identified which are possibly associated with drug resistance, metastasis and can be drug targets.49 In a case, a series of two metastatic and chemo-refractory OGS patients comprehensive molecular profiling was done and molecular targeted therapy was given accordingly. Both the patients did not benefit from the approach.50 Whole exome sequencing, whole transcriptome sequencing, high-density single nucleotide polymorphism array analysis of the tumor and whole exome sequencing of matched germline DNA was done in 59 relapse/refractory paediatric solid tumours to evaluate genome-guided therapy. Out of 59 patients, ten patients were refractory sarcoma [EWS-5, RMS-2, inflammatory myofibroblastic sarcoma-2 and OGS-1]. Actionable mutation with FDA (Unites States Food and Drug Administration) approved drug in adults was seen in four patients while for drugs currently in paediatric trial was seen in five patients. The study showed that multi-dimensional “omics” approach is feasible and can have therapeutic implications.51

The Individualized Therapy for Relapsed Malignancies in Childhood (INFORM) studied 57 patients of relapse paediatric tumours and whole-exome, low-coverage, whole-genome, RNA sequencing, methylation and expression microarray analysis was done. Out of these 57 patients, ten patients of EWS, 5 patients with RMS, 5 patients of OGS and NRSTSwere 3 which were analysable. Eight out of 23 patients had targetable alterations with intermediate or higher prioritization scores.52

In a study of 62 patients with relapsed/refractory paediatric tumours, whole exome sequencing and RNA sequencing were done. Thirteen patients had sarcoma [OGS-7, RMS-4 and EWS-2]. All patients had potentially actionable alterations, which were grouped into targeted therapy, biomarker, risk stratification and diagnostic. Ten patients had actionable alterations for targeted therapy out of which two patients received targeted therapy.53

Comprehensive genomic profiling of 102 advanced/relapse/refractory sarcoma patients were done which included OGS (10), RMS (6) and EWS (3) and actionable mutations were seen in six patients.54 Twenty refractory paediatric sarcoma patients were evaluated for targetable aberrations by array-based expression profiling. All patients had actionable targets. Nine patients received therapy based on actionable alteration and eleven patients did not receive. Median OS and PFS were 8.83 and 6.17 months in the targeted treatment group compared to 4.9 months and 1.7 months in the group which did not receive the targeted therapy, with p values of 0.0014 and 0.0011, respectively. This study showed that patients treated with therapy targeting genetic alterations are likely to benefit.55

In a study of 71 OGS samples from 66 adults and paediatric patients, next-generation sequencing (NGS) was used to identify potentially actionable mutations. Out of these 71 samples, 32 were from metastatic or recurrent lesions. In metastatic/recurrent samples 41.2% had VEGFA whereas the primary site had only 9.7%.56 Amplification of VEGFA has been shown to be a predictor of poor outcomes.57 In the whole cohort, they found genetic changes that can be potentially targeted in 21% and in another 40% they found mutually exclusive VEGFA or PDGFR amplification which can be evaluated as targets. The authors proposed a genetic algorithm classifying approximately 50% of OGS patients eligible for targeted therapy trials.56

Despite encouraging results in identification of targets in refractory paediatric sarcoma, genomic profiling and next-generation sequencing approaches are still experimental.

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