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INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99–not applicable], compared with 117 days (95% CI, 106–143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases.Significance:The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Table from The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets

Supplementary Figure from The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets

Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis.Significance:This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers.See related commentary by Liu and Northcott, p. 2977

Supplementary Figure from FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits

Supplementary Table from FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits

Background: Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. Conclusion: Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis, and further highlights a role for FOXR2 in activating ETS and MAPK signaling. Citation Format: Jessica W. Tsai, Paloma Cejas, Marissa Coppola, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Annarah Charles, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Volker Hovestadt, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3562.

Background: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) expression has been associated with a subset of cancers including CNS and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. Methods: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. Results: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and found FOXR2 to be aberrantly upregulated in 70% of all cancer types, and 8% of all individual tumors. We identified genetic and epigenetic mechanisms that induce its expression, including hypomethylation of a novel promoter in the vast majority (78%) of FOXR2-expressing cases. We demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, using both in vitro and in vivo models. Conclusion: Taken together, this study demonstrates the role of FOXR2 as a potent oncogene across human cancers, and highlights a novel mechanism by which its expression is activated. Citation Format: Jessica W. Tsai, Paloma Cejas, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, J'Ya Hunter, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. FOXR2 is an oncogenic driver across adult and pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5730.

The treatment of relapses from high-risk entities remains a major clinical challenge, thus the desperate need for precision medicine approaches. To serve this need, we have developed the INFORM registry study (INdividualized therapy FOr Relapsed Malignancies in Childhood), which attempts to rapidly generate personalized tumor profiles and identify therapeutic targets in a clinical diagnostic environment for relapse patients. The INFORM study assesses the feasibility of integrating rapid molecular profiling in the clinical management of pediatric cancer patients with progressive or relapsed high-risk malignancies. Whole-exome and low-coverage whole-genome sequencing are being performed on tumor and normal DNA, complemented with matched tumor RNA sequencing (Illumina HiSeq4000), DNA methylation profiling, and gene expression profiling (for outlier gene expression). To date, more than 400 patients were enrolled from >50 centers in seven different countries (Germany, The Netherlands, Switzerland, Austria, Sweden, Finland, and Australia). The average turnaround time from tissue arrival to molecular results is 3 weeks. Actionable targets with at least “borderline” evidence (according to a prioritization score harmonized with the other major pediatric precision oncology programs across Europe) are being identified in ~50% of patients. Based on these findings, several patients were recruited onto ongoing clinical trials, or targeted therapeutics and/or patient-specific peptide vaccines were incorporated into individualized treatment regimes, with first reports of marked responses. Furthermore, we have established a systematic workflow for the reporting of hereditary predisposition, which is detected in ~7% of cases. In 2018, we will start recruiting patients onto several target-defined (entity independent) subtrials of the INFORM2 interventional trial series as well as the complementary counterpart conducted in France, the eSMART trial, which will collectively provide a portfolio of ~10 mechanism-of-action defined, investigator-initiated early phase clinical (combination) trials for pediatric patients at relapse within the European Innovative Therapies for Children with Cancer (ITCC) Consortium. Citation Format: David T. W. Jones, Barbara C.Worst, Elke Pfaff, Cornelis M. Van Tilburg, Gnana Prakash Balasubramanian, Petra Fiesel, Kristian W. Pajtler, Angelika Freitag, Ruth Witt, Andreas E. Kulozik, Felix Sahm, Andreas von Deimling, Angelika Eggert, Uta Dirksen, Peter Lichter, David Capper, Olaf Witt, Stefan M. Pfister. Pediatric precision oncology programs in Germany and Europe [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA11.

A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n = 3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro. To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1×10⁁5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones. Citation Format: Klara M. Giessler, Kortine Kleinheinz, Gnana Prakash Balasubramanian, Daniel Hübschmann, Taronish D. Dubash Rai, Sebastian D. Dieter, Christine Siegl, Christopher M. Hoffmann, Sarah Weber, Raffaele Fronza, Saira Afzal, Manfred Schmidt, Martin Schneider, Alexis Ulrich, Juergen Weitz, Wilko Weichert, Matthias Schlesner, Benedikt Brors, Claudia R. Ball, Hanno Glimm. Genetic subclone heterogeneity of the human colon cancer initiating cell compartment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 910.

Background: Despite substantial progress in treating primary childhood malignancies, relapses from high-risk entities remain a major clinical challenge. The German INFORM study (INdividualized therapy FOr Relapsed Malignancies) is attempting to address this problem using an integrated next-generation sequencing analysis to rapidly generate personalized tumor profiles and identify therapeutic targets. Methods: The INFORM pilot phase assessed the feasibility of integrating rapid molecular profiling into the clinical management of progressive or relapsed high-risk pediatric cancer patients. Whole-exome and low-coverage whole-genome sequencing of tumor and normal DNA was complemented with tumor RNA sequencing (Illumina HiSeq2500, ‘rapid’ mode). This allowed reliable detection of copy-number changes, point mutations, InDels, fusion genes and deregulated gene expression. Identified alterations were prioritized according to tumor biological relevance and potential as an actionable drug target, with results discussed in a weekly molecular tumor board. Results: From Oct 2013 to Jan 2015, 57 patients (average age 13 years) were enrolled from 20 centers throughout Germany. Entities included: high-grade glioma (n = 12), Ewing's sarcoma (n = 11), rhabdomyosarcoma/DSRCT (n = 7), medulloblastoma (n = 5), ependymoma (n = 5), osteosarcoma (n = 4), neuroblastoma (n = 4), and others (n = 9). Tumor tissue was sufficient for analysis of 52 cases. Average turnaround time from tissue arrival to molecular results was 25 days. Actionable targets with at least ‘borderline’ evidence (according to a prioritization score harmonized with other pediatric precision oncology programs across Europe) were identified in 28 patients (49%). The most commonly affected targets included: tyrosine kinases (ALK, n = 5; FGFR, n = 4; MET, n = 4; others, n = 4), the PI3K/mTOR-pathway (PIK3CA, n = 4; PIK3R1, n = 1; TSC2, n = 1), the MAPK pathway (BRAF, n = 1; KRAS, n = 1), the SHH-pathway (PTCH1, n = 3) and cell-cycle control (CCND1/2, n = 4; CDK4, n = 4; CDKN2A/B, n = 3). Based on these findings, targeted therapeutics were incorporated into the therapy regime of several patients (as single experimental treatments or in clinical trials), with anecdotal reports of marked responses. For example, one patient with a previously inoperable myofibroblastic sarcoma is now in complete remission more than 12 months after entering an ALK-inhibitor trial on the basis of our identification of an ALK fusion. Conclusion: A nationwide individualized diagnostic and treatment approach for pediatric cancer patients based on rapid next-generation sequencing is feasible. Our pilot phase results show that actionable targets can be identified in roughly half of the patients. The INFORM registry study has now opened (www.dkfz.de/en/inform/), to collect molecular and clinical data and establish the infrastructure for prospective clinical trials on personalized pediatric oncology. Citation Format: Barbara C. Worst, Cornelis M. van Tilburg, Gnana P. Balasubramanian, Petra Fiesel, David Capper, Miream Boudalil, Stephan Wolf, Sabine Schmidt, Melanie Bewerunge-Hudler, Matthias Schick, Angelika Freitag, Ruth Witt, Lenka Taylor, Andreas von Deimling, Matthias Schlesner, Angelika Eggert, Peter Lichter, David TW Jones, Olaf Witt, Stefan M. Pfister. The INFORM personalized medicine study for high-risk pediatric cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2015-LB-114