Treatment-driven selection of chemoresistant Ewing sarcoma tumors with limited drug distribution
Graphical abstract
Introduction
Ewing sarcoma is a malignancy of the bone and soft tissue of children, adolescents and young adults [1]. In pediatric patients, these tumors - even those (around 50%) classified as high-risk (i.e., those arising in the pelvis or axial skeleton –around 30–40%- and/or with metastases at diagnosis –around 20–30%) - respond well to first-line intensive high-dose chemotherapy [[2], [3], [4]]. For instance, three cycles of high-dose cyclophosphamide, doxorubicin and vincristine induce greater than 90% tumor necrosis in approximately 70% of high-risk patients [2]. In fact, more than 90% of high-risk Ewing sarcoma patients achieve complete remission upon intensive treatment including chemotherapy, radiotherapy and surgery [2,3]. However, more than 50% of patients with metastatic disease at diagnosis and 10–25% of patients with localized disease will relapse within five years of diagnosis [2,4,5]. Rescue pharmacotherapy regimens include camptothecins (irinotecan or topotecan) in combination with cyclophosphamide, temozolomide, or trabectedin [[6], [7], [8]], or taxane-based regimens [9]. Although these treatments actively slow disease, most relapsed tumors respond poorly (or for the short term) to these anticancer drugs, and long term survival for these patients is poor [2,5,10].
The evolution of tumors toward chemoresistance is likely due to the selective pressure of therapies [[11], [12], [13]]. Tumor cells exposed to treatment might be selected to: (i) upregulate oncogenes and downregulate tumor suppressor genes [14]; (ii) alter mitochondrial function to decrease apoptosis [15]; (iii) express drug efflux transporters such as the ATP-binding cassette (ABC) family including ABCB1 (P-glycoprotein/P-gp/MDR1), ABCG2 (Breast Cancer Resistance Protein/BCRP) and ABCC1 (Multidrug Resistance Protein 1/MRP1) [16]; (iv) undergo epithelial to mesenchymal transition [17]; and (v) change tumor dynamics and architecture (blood flow, interstitial fluid pressure, vessel distribution and phenotype, cell density and extracellular matrix) [18]. Some of these mechanisms, especially the expression of drug efflux transporters and changes in tumor dynamics and architecture, may lead to limited drug distribution in tumor cells, which is usually a neglected cause of drug resistance [19].
Whether tumor evolution produces changes in intratumoral drug pharmacokinetics upon intensive treatment in clinically relevant tumor models remains uncharacterized. Therapeutic effect is produced only if the drug can interact with its inner target in the tumor cell at sufficient concentration. However, there has not been in depth in vivo evaluation of anticancer drug distribution in the intracellular target and in other tumor compartments (extracellular and vascular) as it is technically challenging to accomplish [20,21].
To study the compartmental distribution of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in subcutaneous (s.c.) neuroblastoma patient-derived xenografts (PDX), we previously used the microdialysis-tumor homogenate method at the steady state (i.e., at constant drug concentration) in blood [22,23]. In vivo, irinotecan requires the action of the carboxylesterases to be rapidly converted to SN-38 in mice. This metabolite remains active in its lactone form, which predominates at acidic pH and coexists with the carboxylate (inactive) form under a pH-dependent equilibrium [22]. Irinotecan activity is known to be higher in mouse tumor models than in human patients, because there is a higher amount of carboxylesterases in mice [24]. Also, irinotecan/SN-38 biodistribution and activity is restrained by the activity of ABC efflux drug transporters in normal organs, such as the brain, and in tumors [25].
For these studies, we established pairs of Ewing sarcoma PDX models from three pediatric patients. One tumor of each pair was established at diagnosis, and the second was obtained upon relapse or refractory disease progression. We compared the chromosomal profile (copy number alterations; CNA) for each pair. Then, we addressed whether tumor evolution under treatment pressure led to changes in the compartmental intratumoral distribution of SN-38 in vivo, and in ABC transporter expression. We hypothesized that late tumors evolve to be drug resistant and less susceptible to the penetration of anticancer agents.
Section snippets
Establishment of paired Ewing sarcoma PDX models
Tumor biopsies were obtained under an Institutional Review Board-approved protocol at Sant Joan de Déu Hospital (SJD, Barcelona, Spain). Informed consent was obtained from all patients. Samples were obtained from each patient at different stages of treatment, either at diagnosis (early), at advanced (late) relapse or progression refractory stage, in order to establish “early” and “late” patient-matched PDX models. Xenografts were established s.c. in athymic nude mice (Envigo, Barcelona, Spain)
Paired PDX models and patient clinical exposure to irinotecan
We established three pairs of Ewing sarcoma PDX models from biopsies obtained at diagnosis (early) and relapse/progressive refractory (late) stage from three individuals with Ewing sarcoma. Clinical data including EWSR1-FLI1 gene fusions are detailed in Table 1. Patients 1 and 2 achieved complete remission after treatment but relapsed more than one year after diagnosis, off treatment. Patient 3 did not achieve remission and a late biopsy was obtained during refractory progression on treatment.
Discussion
Our studies in patient-matched pairs of PDX models of Ewing sarcoma addressed questions related to changes in drug efficacy and pharmacokinetics within tumor compartments upon tumor evolution. Tumor pairs from patients 1 and 2 after relapse showed the same fusion genes and subtle chromosomic changes compared to early tumor pairs, but were more prone to become chemoresistant and less susceptible to intracellular drug distribution. The patient-matched tumor pair obtained from primary refractory
Conclusion
In summary, using a unique set of clinically relevant PDX pairs established at early and late tumor stages, we have identified that drug concentration in Ewing sarcoma tumor cells becomes restricted during tumor evolution under treatment pressure. We observed this in two cases in which CNAs in early and late xenografts were similar, suggesting a common clonal origin. Our results should be taken cautiously due to the low number of cases, but they might be clinically relevant to predict response
Declaration of Competing Interest
None.
Acknowledgements
The work at Hospital Sant Joan de Deu was supported by associations of parents and families of children with cancer. The work of EdA was supported by the AECC Scientific Foundation (GCB13-1578), ISCIII-FEDER (PI14/01466 and PI17/00464), CIBERONC (CB16/12/00361), Asociación Pablo Ugarte and Fundación María García Estrada. The work of JM was supported by the AECC Scientific Foundation (GCB13-1578) and Asociación Pablo Ugarte. The work of AMC was supported by grants from the AECC Scientific
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