Generation of pediatric liver cancer patient–derived xenograft platforms for pediatric liver cancer: A critical stage in the development of anticancer treatments
Authors: Gregory Tiao, James Geller, Nikolai A. Timchenko
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on Page 1121 Hepatoblastoma (HBL) is the most common form of pediatric liver cancer, and there is an urgent need for discovery of the mechanisms of HBL and for the development of novel, less toxic therapeutic approaches to treat and cure children with existing cancer. Investigations of HBL have intensified during recent years; however, few have addressed the large‐scale global alterations of gene expression which occur. Luo et al. investigated seven HBL samples and compared them with 37 hepatocellular carcinoma (HCC) samples, demonstrating fibronectin, delta‐like 1 homolog, transforming growth factor‐β, and mitogen‐inducible gene 6 to be highly expressed in HBL.1 A second global analysis of HBL samples performed by a multi‐investigator team revealed the highly significant role of Wnt‐β‐catenin signaling in HBL and identified a 16‐gene signature associated with aggressive HBL.2 This report and a report by Cairo's group provided evidence that HBL has stem cell–like signatures.2 A fourth recent article reports exome sequencing of 47 HBL samples.4 The interesting results of this study showed that HBL is a very simple tumor genetically, with 2.9 mutations per tumor, mainly in the β‐catenin (CTNNB1) and nuclear factor erythroid 2–like 2 (NFE2L2) genes.4 In summary, investigations to date suggest a high reliance on RNA, micro‐RNA, protein, and epigenetic biology driving HBL pathogenesis, rather than gene mutational burden. Given such complex biology as well as heterogeneity of pediatric liver cancer, it becomes clear that the elucidation of therapeutically targetable mechanisms of HBL will take significant amounts of time and energy. Preclinical studies with animal models are usually performed prior to clinical trials. While several animal models are well developed for HCC, limited genetically engineered models have been described that recapitulate the features of HBL. One of these models presents activation of β‐catenin, which is sufficient for the development of HBL and HCC.5 However, the development of tumors appeared in these mice when they reached adult age, 26 weeks.5 Given the limitations of genetically engineered animal models of HBL, the development and use of xenograft platforms are the most productive and efficient ways for the preclinical exploration of drugs for possible treatment of pediatric liver cancers. In this issue of Hepatology, Cairo's group presents the establishment of preclinical platforms of pediatric liver cancer patient–derived xenografts (PLC‐PDXs), analyses of the tumors, and treatments of these cancers with known therapeutic interventions.6 It is important to note that this work is a continuation of Dr. Cairo's key contributions to the studies of HBL.2 In this article, the authors collected 58 liver tumor specimens from 51 children between the years 2010 and 2015 and grafted them into the interscapular region of young athymic nude mice. They established PDX models from 20 HBLs, one translational cell liver tumor, one HCC, and two malignant rhadboid tumors. These tumor samples were from patients who underwent preoperative chemotherapy. Among established PLC‐PDXs, the authors generated one PDX derived from α‐fetoprotein–negative, very aggressive HBL, which is observed in <1% of pediatric liver tumors.7 These PDX models were further used for the investigation of effects of known and new therapeutic options on liver growth and tumor features. Initially, the authors demonstrated that the grafted tumors did not affect liver functions of recipient mice. One of the key concerns in generation of PLC‐PDXs is if the grafted liver tumors recapitulate the histology and genomic profiling of the tumors of origin. Histological examination of the generated PLC‐PDX models showed that they recapitulate the tumor cell morphology and tissue organization of the tumors of origin at different passages. Because the most frequent mutations in HBL are related to the CTNNB1 and NFE2L2 genes, the authors further compared mutations in these genes in PLC‐PDX platforms with mutations in the tumors of origin. These experiments demonstrated that PLC‐PDXs reflect genetic profiles of the tumor of origin. Examination of 58 genes by exon sequencing in three PLC‐PDXs revealed that only three genes, CTNNB1, NFE2L2, and integrase interactor 1/SWI/SNF‐related, matrix‐associated, actin‐dependent regulator of chromatin, subfamily B, member 1 (INI1/SMARCB1), have been found to be mutated. The main characteristics of HBL are tumor growth and secretion of α‐fetoprotein. Examination of these parameters in PCL‐PDXs revealed that these two parameters were preserved in the established PCL‐PDXs at levels close to those observed in patients. In general, these investigations of the features of generated PLC‐PDXs showed that they recapitulate the pathophysiological and molecular properties of the tumors of origin. In the next set of experiments, Nicolle et al. examined the response of PLC‐PDXs to standard treatments of liver cancer and to new combinations of drugs. These studies provided several interesting observations. First, the authors showed that doxorubicin alone did not show effects on tumor growth in all examined PLC‐PDXs. Doxorubicin also did not show a cooperative effect if administrated with cisplatin. PLC‐PDX platforms showed that cisplatin also had weak effects on tumor growth. In addition to these compounds, the authors tested the effects of irinotecan, temozolomide, temsirolimus, sirolimus, nefopam, sorafenib, crizotinib, and paclitaxel on the tumor growth of established PDXs. Several combinations of these compounds were also tested. In these studies, the authors found that irinotecan alone or in combination with temozolomide strongly induced tumor suppression and reduction of α‐fetoprotein secretion, while combinations with other compounds showed very weak to no growth inhibition in these PDX models. Interestingly, the authors found that a 4‐fold reduction (from 40 mg/kg to 10 mg/kg) dose of the irinotecan/temozolomide combination had the same inhibitory effect on the tumor as the higher dose. This observation is highly significant as it provides support for the reduction of toxicity of the investigated compounds when applied in patients. It is important to note that, although studies with PLC‐PDX tools show the effects of compounds in vivo on human tumors, the influence of a healthy mouse body may differ from the effects of a human environment, which is commonly altered by liver cancer disease. Another weakness of the established PDXs is that tumors were grafted in mouse interscapular regions that reflect the physiology of metastasis rather than intrahepatic tumor. Despite these problems, an established large panel of pediatric liver cancer xenografts is arguably the best system for preclinical examination of drugs to treat pediatric liver cancer. In addition, PLC‐PDXs are powerful tools for investigations of the mechanisms of liver cancer. Figure 1 summarizes a scope of questions that might be addressed using PLC‐PDXs.Figure 1: The publication by Nicolle and colleagues describes the powerful PLC‐PDX tools that might be used in the search for optimal therapeutic approaches and for investigations of mechanisms of pediatric liver cancer.In conclusion, the work by Nicolle et al. provides a significant advance in the development of preclinical tools necessary for the advancement of novel therapeutic and biologic investigations of pediatric liver cancer, especially for chemoresistant clones. The search for a common therapy for pediatric liver tumor remains complicated by tumor heterogeneity, however.9 Therefore, future investigations into the development of PDXs should be coordinated with elucidation of the mechanisms of heterogeneity of pediatric liver tumor, with elucidation of mechanisms of chemoresistance, and with elucidation of pathways, alterations of which are both inherent to the tumor as well as mediated by a chemotherapeutic effect. These future studies should improve the application and generalizability of PLC‐PDX models as tools for preclinical studies.