ITCC-P4: Molecular characterization and multi-omics analysis of pediatric patient tumor and Patient-Derived Xenograft (PDX) models for preclinical model selection

Cancer persists as one of the prevailing causes of death in children and adolescents aged 0 to 19 years. There remains to be an unmet need for identification of therapeutic biomarkers and better treatment interventions for these patients. Advancements in state-of-the-art molecular profiling techniques have resulted in better understanding of pediatric cancers and their driver events. It has become apparent that pediatric malignancies are significantly more heterogeneous than previously thought as evidenced by the number of novel entities and subtypes that have been identified with distinct molecular and clinical characteristics. For most of these newly recognized entities there are currently extremely limited treatment options available. Unfortunately, there is also a lack of compiled and consistently analysed molecular data available, along with limited data of characterization and documentation of patient-derived models and/or genetic mouse models from high-risk pediatric tumors. Both my studies fall under the “Innovative Therapies for Children with Cancer Pediatric Preclinical Proof-of-concept Platform” (ITCC-P4) consortium which is an international collaboration between different European academic institutes, several partnering pharmaceutical companies and three contract research organizations. The two studies aim to shed light on identification of potential promising treatment options that specifically match the patient’s specific molecular tumour characteristics and the patient’s genetic data. Genetic information at the molecular level from pediatric tumors in relapsed patients has contributed to advancing our understanding of disease progression and treatment resistance. The first study overall aims to establish a sustainable platform of >400 molecularly well- characterized PDX models of high-risk pediatric cancers, including the analysis of their original tumors and matching controls. This will enable the selection of PDX models for in vivo testing of novel mechanism-of-action based treatments. Hence, facilitating the prioritization of pediatric drug development and clinical stratification of patients across entities. In a first batch, 251 models were fully characterized, including 180 brain and 71 non- brain PDX models, representing 112 primary models, 93 relapse, 42 metastasis and 4 progressions under treatment models. Using low-coverage whole-genome and deep whole exome sequencing, complemented with total RNA sequencing and methylation analysis, the aim was to define genetic features in the ITCC-P4 PDX cohort and assess the molecular fidelity of PDX models compared to the original tumor. Based on DNA methylation profiling 43 different tumor subgroups within 18 cancer entities were included. Mutational landscape analysis identified key somatic and germline oncogenic drivers where Ependymoma PDX models displayed the C11orf95-RELA fusion event, YAP1, C11orf95 and RELA structural variants. Medulloblastoma models were driven by MYCN, TP53, GLI2, SUFU and PTEN. High-grade glioma samples showed TP53, ATRX, MYCN and PIK3CA somatic SNVs, along with focal deletions in CDKN2A in chromosome 9. Neuroblastoma models were enriched for ALK SNVs and/or MYCN focal amplification, ATRX SNVs and CDKN2A/B deletions. Sarcoma models displayed characteristic alterations with PAX3-FOXO1 fusions detected in embryonal rhabdomyosarcoma, along with TP53, CDKN2A, NRAS SNVs, NCOA1 gains, NF1 and CDK4 SVs. Ewing sarcoma PDX models displayed the defining EWSR1-FLI1 gene fusion in most cases, along with two rarer cases of EWSR1-ERG and EWSR1-FEV observed in the cohort. Osteosarcomas were defined by highly unstable genomes with large chromosomal alterations, TP53 and RB1 tumor suppressor genes were frequently altered and ATRX loss and MYC gains were observed. Additional sarcomas such as clear cell sarcoma of the kidney showed CDKN2A loss, MYC gain, NF1 loss, TP53 mutations, while Synovial sarcoma models were driven by SSX gene fusions and alterations. Large chromosomal aberrations (deletions, duplications) detected in the PDX models were concurrent with molecular alterations frequently observed in each tumor type –isochromosome 17 was detected in five medulloblastoma models, while deletion of chromosome arm 1p or gain of parts of 17q in neuroblastomas which correlate with tumor progression. Tumor mutational burden across entities and copy number analysis was performed to identify allele-specific copy number events in tumor-normal pairs. Clonal evolution of somatic variants was not only found in certain PDX-tumor pairs but also between disease states. Across the 16 serial model cases, discordance in targetable SNV, SV and CNV, alterations were observed in later disease progressed states compared to the primary models. The multi-omics approach in this study provides insight into the mutational landscape and patterns of the PDX models thus providing an overview of molecular mechanisms facilitating the identification and prioritization of oncogenic drivers and potential biomarkers for optimal treatment. The second study was a Target Actionability Review on replication stress. Detrimental long-term side effects due to chemotherapy drastically affect the lives of patients under treatment, hence there is an urgent need to identify novel target driven therapies. Decades of published data provide evidence for targeting replication stress therapeutically. Hence, in this study, we evaluated specific targets within the replication stress response (RSR) pathway. A comprehensive, well-structured, and critically evaluated overview of literature related to replication stress across 16 pediatric solid malignancies was generated. The methodology focuses on the systemic extraction and structured evaluation of replication stress as a target. This aims to align targeted anti- cancer therapeutic interventions with specific cancer subtypes based on clinical studies. ATR, ATM, PARP, WEEI were observed to represent the most promising targets either using single agents or in combination with chemotherapy or radiotherapy. Evidence on CHK1 and DNA-PK although limited, showed potential to further investigate these promising targets over broader tumor types. The collective data and results from both studies, the “ITCC-P4: Molecular characterization and multi-omics analysis of Patient-Derived Xenograft (PDX) models from high-risk pediatric cancer” and the “Target actionability review on replication stress”, can be explored further on the interactively designed R2 platform, once users create an account to gain access to the cohort data. (https://r2-itcc-p4.amc.nl/)

iTReX: Interactive exploration of mono- and combination therapy dose response profiling data

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application’s ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at (https://itrex.kitz-heidelberg.de).Peer reviewe

Target Actionability Review: a systematic evaluation of replication stress as a therapeutic target for paediatric solid malignancies

Background: Owing to the high numbers of paediatric cancer-related deaths, advances in therapeutic options for childhood cancer is a heavily studied field, especially over the past decade. Classical chemotherapy offers some therapeutic benefit but has proven long-term complications in survivors, and there is an urgent need to identify novel target-driven therapies. Replication stress is a major cause of genomic instability in cancer, triggering the stalling of the replication fork. Failure of molecular response by DNA damage checkpoints, DNA repair mechanisms and restarting the replication forks can exacerbate replication stress and initiate cell death pathways, thus presenting as a novel therapeutic target. To bridge the gap between preclinical evidence and clinical utility thereof, we apply the literature-driven systematic target actionability review methodology to published proof-of-concept (PoC) data related to the process of replication stress. Methods: A meticulous PubMed literature search was performed to gather replication stress-related articles (published between 2014 and 2021) across 16 different paediatric solid tumour types. Articles that fulfilled inclusion criteria were uploaded into the R2 informatics platform [r2.amc.nl] and assessed by critical appraisal. Key evidence based on nine pre-established PoC modules was summarised, and scores based on the quality and outcome of each study were assigned by two separate reviewers. Articles with discordant modules/scores were re-scored by a third independent reviewer, and a final consensus score was agreed upon by adjudication between all three reviewers. To visualise the final scores, an interactive heatmap summarising the evidence and scores associated with each PoC module across all, including paediatric tumour types, were generated. Results and conclusions:: 145 publications related to targeting replication stress in paediatric tumours were systematically reviewed with an emphasis on DNA repair pathways and cell cycle checkpoint control. Although various targets in these pathways have been studied in these diseases to different extents, the results of this extensive literature search show that ATR, CHK1, PARP or WEE1 are the most promising targets using either single agents or in combination with chemotherapy or radiotherapy in neuroblastoma, osteosarcoma, high-grade glioma or medulloblastoma. Targeting these pathways in other paediatric malignancies may work as well, but here, the evidence was more limited. The evidence for other targets (such as ATM and DNA-PK) was also limited but showed promising results in some malignancies and requires more studies in other tumour types. Overall, we have created an extensive overview of targeting replication stress across 16 paediatric tumour types, which can be explored using the interactive heatmap on the R2 target actionability review platform [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1]

iTReX: Interactive exploration of mono- and combination therapy dose response profiling data

High throughput screening methods, measuring the sensitivity and resistance of tumor cells to drug treatments have been rapidly evolving. Not only do these screens allow correlating response profiles to tumor genomic features for developing novel predictors of treatment response, but they can also add evidence for therapy decision making in precision oncology. Recent analysis methods developed for either assessing single agents or combination drug efficacies enable quantification of dose-response curves with restricted symmetric fit settings. Here, we introduce iTReX, a user-friendly and interactive Shiny/R application, for both the analysis of mono- and combination therapy responses. The application features an extended version of the drug sensitivity score (DSS) based on the integral of an advanced five-parameter dose-response curve model and a differential DSS for combination therapy profiling. Additionally, iTReX includes modules that visualize drug target interaction networks and support the detection of matches between top therapy hits and the sample omics features to enable the identification of druggable targets and biomarkers. iTReX enables the analysis of various quantitative drug or therapy response readouts (e.g. luminescence, fluorescence microscopy) and multiple treatment strategies (drug treatments, radiation). Using iTReX we validate a cost-effective drug combination screening approach and reveal the application’s ability to identify potential sample-specific biomarkers based on drug target interaction networks. The iTReX web application is accessible at (https://itrex.kitz-heidelberg.de).Peer reviewe

The genomic landscape of pediatric renal cell carcinomas

Pediatric renal cell carcinomas (RCC) differ from their adult counterparts not only in histologic subtypes but also in clinical characteristics and outcome. However, the underlying biology is still largely unclear. For this reason, we performed whole-exome and transcriptome sequencing analyses on a cohort of 25 pediatric RCC patients with various histologic subtypes, including 10 MiT family translocation (MiT) and 10 papillary RCCs. In this cohort of pediatric RCC, we find only limited genomic overlap with adult RCC, even within the same histologic subtype. Recurrent somatic mutations in genes not previously reported in RCC were detected, such as in CCDC168, PLEKHA1, VWF, and MAP3K9. Our papillary pediatric RCCs, which represent the largest cohort to date with comprehensive molecular profiling in this age group, appeared as a distinct genomic subtype differing in terms of gene mutations and gene expression patterns not only from MiT-RCC but also from their adult counterparts

Target Actionability Review: a systematic evaluation of replication stress as a therapeutic target for paediatric solid malignancies

Background: Owing to the high numbers of paediatric cancer-related deaths, advances in therapeutic options for childhood cancer is a heavily studied field, especially over the past decade. Classical chemotherapy offers some therapeutic benefit but has proven long-term complications in survivors, and there is an urgent need to identify novel target-driven therapies. Replication stress is a major cause of genomic instability in cancer, triggering the stalling of the replication fork. Failure of molecular response by DNA damage checkpoints, DNA repair mechanisms and restarting the replication forks can exacerbate replication stress and initiate cell death pathways, thus presenting as a novel therapeutic target. To bridge the gap between preclinical evidence and clinical utility thereof, we apply the literature-driven systematic target actionability review methodology to published proof-of-concept (PoC) data related to the process of replication stress. Methods: A meticulous PubMed literature search was performed to gather replication stress-related articles (published between 2014 and 2021) across 16 different paediatric solid tumour types. Articles that fulfilled inclusion criteria were uploaded into the R2 informatics platform [r2.amc.nl] and assessed by critical appraisal. Key evidence based on nine pre-established PoC modules was summarised, and scores based on the quality and outcome of each study were assigned by two separate reviewers. Articles with discordant modules/scores were re-scored by a third independent reviewer, and a final consensus score was agreed upon by adjudication between all three reviewers. To visualise the final scores, an interactive heatmap summarising the evidence and scores associated with each PoC module across all, including paediatric tumour types, were generated. Results and conclusions:: 145 publications related to targeting replication stress in paediatric tumours were systematically reviewed with an emphasis on DNA repair pathways and cell cycle checkpoint control. Although various targets in these pathways have been studied in these diseases to different extents, the results of this extensive literature search show that ATR, CHK1, PARP or WEE1 are the most promising targets using either single agents or in combination with chemotherapy or radiotherapy in neuroblastoma, osteosarcoma, high-grade glioma or medulloblastoma. Targeting these pathways in other paediatric malignancies may work as well, but here, the evidence was more limited. The evidence for other targets (such as ATM and DNA-PK) was also limited but showed promising results in some malignancies and requires more studies in other tumour types. Overall, we have created an extensive overview of targeting replication stress across 16 paediatric tumour types, which can be explored using the interactive heatmap on the R2 target actionability review platform [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1]