The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) ...and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.
Background
The development of high‐throughput drug screening (HTS) using primary cultures provides a promising, clinically translatable approach to tailoring treatment strategies for patients with ...cancer. However, this has been challenging for solid tumors because of often limited amounts of tissue available. In most cases, in vitro expansion is required before HTS, which may lead to overgrowth and contamination by non‐neoplastic cells.
Methods
In this study, hematoxylin and eosin staining and immunohistochemical staining were performed on 129 cytopathology cases from 95 patients. These cytopathology cases comprised cell block preparations derived from primary tumor specimens or patient‐derived xenografts as part of a pediatric precision oncology trial. Cytopathology cases were compared with the morphology and immunohistochemical staining profile of the original tumor. Cases were reported as tumor cells present, equivocal, or tumor cells absent. The HTS results from cytopathologically validated cultures were incorporated into a multidisciplinary tumor board report issued to the treating clinician to guide clinical decision making.
Results
On cytopathologic examination, tumor cells were present in 77 of 129 cases (60%) and were absent in 38 of 129 cases (29%), whereas 14 of 129 cases (11%) were equivocal. Cultures that contained tumor cells resembled the tumors from which they were derived.
Conclusions
Cytopathologic examination of tumor cell block preparations is feasible and provides detailed morphologic characterization. Cytopathologic examination is essential for ensuring that samples submitted for HTS contain representative tumor cells and that in vitro drug sensitivity data are clinically translatable.
Routine cytopathology is able to establish the presence or absence of tumor cells in primary cultures derived from patients with pediatric cancer. Cytopathologic examination ensures the clinical relevance of in vitro drug sensitivity profiling in a pediatric precision oncology platform.
Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid ...high‐throughput drug screening (HTS) and patient‐derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high‐risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high‐risk pediatric cancer patients.
Synopsis
A precision diagnostic platform integrating genomics and transcriptomics with drug testing of patient's primary tumor cells in high throughput drug screening (HTS) and patient‐derived xenograft (PDX) was established to improve identification of therapies in high‐risk pediatric cancer patients.
Treatment options could be identified for 70% of patients across the four‐part platform.
HTS provided orthogonal proof of drug efficacy suggested by molecular analyses and identified many new drug responses without prior molecular hallmarks.
Effective treatments were observed in more than half of PDX models.
There was a strong correlation between HTS and PDX results, and the clinical responses in patients.
A precision diagnostic platform integrating genomics and transcriptomics with drug testing of patient's primary tumor cells in high throughput drug screening (HTS) and patient‐derived xenograft (PDX) was established to improve identification of therapies in high‐risk pediatric cancer patients.
Abstract
The survival rate of acute myeloid leukemia (AML) patients undergoing standard chemotherapy remains poor due to disease relapse. The main cause of relapse is the failure of chemotherapeutic ...drugs to eradicate self-renewing leukemic stem cells (LSCs) (Cancers 2017;9:74-97). Because of their intrinsic drug resistance, LSCs are extremely difficult to target by single-gene therapies. MicroRNAs (miRNAs), a class of small noncoding RNAs capable of regulating multiple target genes, represent a promising therapeutic target for elimination of LSCs. While miRNA-101 (miR-101) has been identified as a tumor suppressor in several types of cancers including prostate (Science 2008;322:1695-99) and liver cancer (Cancer Res 2009;69:2623-29), its expression and functional role in AML remains unknown. In this study, we uncover that miR-101 plays a crucial role in leukemogenesis through modulation of key epigenetic and signaling pathways activated in LSCs of mixed-lineage leukemia (MLL)-rearranged AML. Our data showed that overexpression of miR-101 in murine pre-LSCs inhibited cell proliferation in vitro and delayed leukemogenesis in vivo, confirming a tumor-suppressor function of miR-101 in MLL-AF9-induced AML, a highly aggressive leukemia. To assess the therapeutic value of miR-101, we next examined the antitumor function of miR-101 in a xenograft model of human MLL-AF9 AML. Briefly, miR-101 was overexpressed in human MLL-AF9 AML cell line, MOLM-13, that was then transplanted into NOD-scid IL2Rgammanull (NSG) mice followed by in vivo bioluminescence imaging to monitor engraftment of human leukemic cells. Our result revealed a significantly lower engraftment in the xenograft mouse model carrying miR-101-overexpressing leukemic cells compared to control mice. This suggests that inhibition of tumor suppressor miR-101 is essential in the initiation of an aggressive form of human AML. To investigate the potential mechanism by which miR-101 impairs leukemogenesis, we examined key oncogenic pathways regulated by miR-101 by performing luciferase assays and Western blotting. Our data showed that miR-101 inhibited leukemogenesis via directly targeting Enhancer of zeste homolog 2 (EZH2), a crucial epigenetic regulator in leukemia maintenance, as well as key components of Wnt/β-catenin signaling, an oncogenic pathway necessary for LSC self-renewal in AML. Furthermore, immunofluorescence analysis revealed a significant reduction in active β-catenin in the nucleus of miR-101-overexpressing leukemic cells, indicating a direct regulation of miR-101 in β-catenin activity. Collectively, these findings reveal a novel role for miR-101 in leukemia progression that may be used to develop a targeted therapy for AML treatment.
Citation Format: Estrella Gonzales-Aloy, Dylan G. Grebert-Wade, Jenny Y. Wang. Tumor suppressor miRNA-101 modulates leukemogenesis by targeting the EZH2/Wnt/β-catenin signaling pathways abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 484.
Abstract
Molecular genomics analyses aim to identify subsets of patients harboring actionable aberrations as a pathway to improved targeted treatment selection. However, recent pan-cancer analyses of ...the molecular landscape of pediatric cancers1,2 have emphasized the stark contrast with adult cancers, with low mutation rates, distinct mutated genes and a prevalence of structural rearrangements suggesting that genomic analyses alone have limitations for translation into clinical benefit. The Zero Childhood Cancer (ZCC) program aims to assess the feasibility of precision medicine to identify targeted therapeutic agents for patients with high-risk (HR) pediatric malignancies (expected survival <30%). We combine comprehensive molecular profiling analysis whole genome sequencing (tumor, germline DNA), deep sequencing of a 386 cancer associated gene panel, whole transcriptome (RNASeq), methylation profiling with in vitro high-throughput drug screening (124 compound library, single agent) and patient-derived xenograft (PDX) drug efficacy testing. Results are curated and recommendations made by a national Multidisciplinary Tumor Board. Recommendations consist of targeted therapy, change of diagnosis or genetics referral for a germline cancer predisposition gene mutation. The national multicenter prospective trial (PRISM) opened in September 2017 at all 8 pediatric oncology centers around Australia, following the successful completion of a 2-year pilot feasibility study. PRISM has enrolled 131 patients to date (35% central nervous system tumors, 29% sarcoma, 13% leukemias/lymphomas, 6% neuroblastoma, 17% other rare or unknown cancers). The unique ZCC testing platform has resulted in at least one recommendation being issued for 67% of patients. Fifteen % of patients have a reportable germline cancer predisposition. We have developed an analytical pipeline to interrogate and cross-validate the full range of variants, structural abnormalities and mutational signatures identified in pediatric cancers and incorporate the molecular data with in vitro and in vivo drug sensitivity data where possible. The highest yield of reportable variants is derived from the integrated analysis of WGS and RNASeq; unique to ZCC compared to other pediatric precision medicine programs internationally. ZCC demonstrates the feasibility of a comprehensive precision medicine platform to identify treatment recommendations in HR pediatric cancer patients. The national trial is planned to run for 3 years, recruiting ~400 patients. In addition, ZCC is partnering nationally and internationally to conduct parallel research studies in immunoprofiling, liquid biopsy, psychosocial impact of precision medicine, health economics and health implementation. 1. Gröbner et al. Nature. 2018; 555(7696):321-327. 2. Ma et al. Nature. 2018; 555(7696):371-376.
Citation Format: Emily V. Mould, Loretta Lau, Greg Arndt, Paulette Barahona, Mark J. Cowley, Paul Ekert, Tim Failes, Jamie Fletcher, Andrew Gifford, Dylan Grebert-Wade, Michelle Haber, Alvin Kamili, Amit Kumar, Richard B. Lock, Glenn M. Marshall, Chelsea Mayoh, Murray Norris, Tracey O'Brien, Dong Anh Khuong Quang, Patrick Strong, Alexandra Sherstyuk, Toby Trahair, Maria Tsoli, Katherine Tucker, Meera Warby, Marie Wong, Jinhan Xie, David S. Ziegler, Vanessa Tyrrell. Zero Childhood Cancer: A comprehensive precision medicine platform for children with high-risk cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3111.
Abstract
Despite the increase in overall child cancer survival rates, pediatric malignancies such as high-risk neuroblastoma, high-risk leukemias (including MLL-translocated infant ALL), and ...aggressive brain tumors (including DIPG) remain refractory to current multimodal therapies. We have been developing new treatment approaches for these aggressive childhood cancers by (i) utilizing novel targeted therapies either alone or combined with other new agents or established chemotherapeutic drugs, and (ii) by developing new drugs that target key pathways in these child cancers.
In neuroblastoma, we have targeted polyamines, showing that combined inhibition of polyamine synthesis by the ODC1 inhibitor DFMO, and of polyamine uptake using the small-molecule drug AMXT 1501, is highly effective at inhibiting tumor growth in Th-MYCN transgenic mice. This combination also shows great efficacy in preclinical models of DIPG, and clinical trials for these diseases are now being planned. We are also targeting metabolism of arginine, the precursor of ornithine, using the pegylated-recombinant arginase BCT-100, which significantly delays tumor development and prolongs survival of neuroblastoma-prone Th-MYCN mice. We have further shown that combining BCT-100 with either DFMO or conventional chemotherapy results in increased survival benefit.
CBL0137 is a nontoxic novel anticancer drug currently in phase I trial for adult refractory and relapsed cancers. CBL0137 destabilizes nucleosomes and traps histone chaperone FACT into chromatin, thereby modulating several anticancer mechanisms. We have shown that CBL0137 is effective in mouse models of neuroblastoma, MLL-rearranged leukemia, and DIPG, and that its action is potentiated by the HDAC inhibitor, panobinostat. Moreover, we have developed OT-82, a novel nontoxic NAMPT inhibitor with impressive anticancer activity against mouse models of high-risk childhood ALL, potentiating standard-of-care drugs, and showing similar efficacy as the three-drug induction-type treatment used for pediatric ALL.
In addition, for all Australian children with high-risk malignancies, we have developed the Zero Childhood Cancer national precision medicine program. ZERO utilizes whole-genome and whole-transcriptome sequencing, methylation profiling, and where possible, in vitro and in vivo drug testing. To date (July 2019), 74% of 207 patients on the national clinical trial have received a Multidisciplinary Tumor Board recommendation (therapy, germline referral, or change of diagnosis), and of 25 patients with evaluable response data thus far who have received the ZERO recommended therapy, a significant proportion have had a complete response, partial response, or maintained stable disease. Moreover, early experience with drug efficacy studies suggests these data may corroborate genomic therapeutic recommendations and may also identify unanticipated active therapeutics.
Citation Format: Michelle Haber, Laura Gamble, Lin Xiao, Ruby Pandher, Klaartje Somers, Jayne Murray, Aaminah Khan, Denise Yu, Laura Franshaw, Mark R. Burns, Maria Tsoli, Anahid Ehteda, Anthony Cesare, Aisling O’Connor, Francis Mussai, Carmela de Santo, Paul Cheng, Lioubov Korotchkina, Katerina Gurova, Vanessa Tyrrell, Emily Mould, Loretta Lau, Dong Anh Khuong Quang, Chelsea Mayoh, Greg Arndt, Paulette Barahona, Tim Failes, Jamie Fletcher, Noemi Fuentes- Bolanos, Marie-Emilie Gauthier, Andrew Gifford, Dylan Grebert-Wade, Alvin Kamili, Amit Kumar, Sumanth Nagabushan, Tracey O’Brien, Patrick Strong, Alexandra Sherstyuk, David Thomas, Toby Trahair, Katherine Tucker, Meera Warby, Marie Wong, Jinhan Xie, Kathryn Evans, Richard Lock, Olga B. Chernova, Michelle Henderson, Andrei V Gudkov, Paul Ekert, Mark J. Cowley, Glenn M. Marshall, David S. Ziegler, Murray D. Norris. Molecular targeted therapies and precision medicine for children with neuroblastoma and other refractory malignancies abstract. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA13.
Abstract
Brain tumours represent the most common solid tumour of childhood and result in significant morbidity and mortality. The Zero Childhood Cancer national child precision medicine program aims ...to identify targeted therapeutic agents for high-risk paediatric malignancies (expected survival <30%) including brain tumours. Here we will report on the Pilot Feasibility Study (TARGET) and the initial experience of the National Clinical Trial (PRISM), which opened in September of 2017. A total of 200 patients have been enrolled, 59 in the pilot phase (TARGET) and 141 in the National study (PRISM) out of which 77 patients (38.5%) had CNS malignancies, of which 64 cases have completed curation. Molecular analysis of these cases identified actionable molecular aberrations in 47 patients (73.4%). Ten cases (15.6%) had a reportable germline cancer predisposition variant. Overall, the most common aberrant genetic changes observed include TP53 mutations, CDKN2A/B biallelic loss, PDGFRA over-expression mainly in the presence of amplification, and fusions containing either NTRK or BRAF. In 2 cases, the somatic genomic findings changed the primary diagnosis. Fresh tissue collection permitted in vitro high throughput screening (HTS) (120 single agents) in 32/69 (46.3%) of cases with additional cultures currently under development. Hits were identified in 2 cultures and recommendations were made. Four PDX models from successful primary cultures were established where single and combination drug efficacy studies have been performed based on recommendations made from molecular profiling or HTS analysis. Currently multiple PDX models are under evaluation either from successful primary cultures or direct intracranial injection of biopsies. In this study we will present an overview of the molecular and preclinical platforms and their impact on the management of paediatric patients with aggressive brain tumours.
PDF
