Background and Aims

Childhood acute lymphoblastic leukemia (ALL) genome landscapes indicate relapsed cancer often arise from subclonal outgrowths. However, the impact of clonal evolution on response to targeted therapy and the actionable proteome is not known. Here, we interrogate ALL proteogenome dynamics in patient-matched diagnosis (Dx), relapse (R) specimens, specifically to understand how cancer-driving and potentially targetable lesions persist or differentiate through disease progression.

Methods

We sourced patient-matched Dx,R specimens (n=11 patients, BC Children’s Hospital (BCCH)) or non-cancer pediatric specimens (n=3 donors). We explored the mutational landscape via targeted, pediatric cancer-focused next generation sequencing (NGS). Public whole-genome NGS data was also analyzed for additional matched ALL samples (n=69 patients, St. Jude Hospital). Proteomes were performed on BCCH specimen and analyzed for equivalence across the whole proteome and within 269 pediatric cancer-driving gene products. Finally, viably-frozen primary samples were treated in vitro with targeted agents based on targets identified via NGS or proteome analysis.

Results

NGS analysis indicated retention of Dx variants in R samples for 56 of 80 (70%) patients. Viably-frozen matched Dx-R/R1-R2 samples (n=6 patients) showed poorly selective but highly correlated responses to four NGS variant-targeted drugs. Patient-matched samples (n=11 patients) showed the highest equivalence of protein expression. Versus normal samples, 33 proteins (of defined cancer-drivers) were significantly changed in disease samples; for these proteins, expression was similar in patient-matched Dx,R samples. Whole-proteome analysis (n=11 patients) identified PARP1 as a potential pan-ALL target and viably-frozen ALL specimens (n=18 patients) treated ex vivo showed heightened sensitivity to two PARP1/2 inhibitors relative to non-cancer samples (n=5 donors).

Conclusions

Changes in variant frequencies mark disease evolution. But, proteomes, targetable lesions and sensitivity to targeted agents remain relatively stable in patient-matched progression samples. Thus, initiation of a precision medicine workflow when pediatric ALL is first diagnosed has the potential to identify disease sensitivities that are likely to persist at disease relapse.

Background and Aims

The implementation of liquid biopsies into clinical practice is well underway for adult patients, but its application to paediatric cancer patients lags behind. Minimally invasive molecular profiling could provide a powerful platform to guide clinical decision-making and deliver precision treatments, particularly at the time of relapse when tumour biopsy may not be possible. However, paediatric cancers have significantly different mutation profile than adult patients and paediatric-tailored profiling methods are needed.

Methods

To fill in this gap we have developed a clinically relevant pan-paediatric solid tumour NGS capture panel optimised for cell free DNA (cfDNA). We have validated the method to clinical reporting standards with a limit of detection of 0.125% variant allele frequency and combined it with low coverage whole genome sequencing of the same library to inform on genome wide copy number changes.

Results

We applied this method to time matched cfDNA (from blood) and tissue biopsy samples from 250 paediatric patients with a range of solid tumours at relapse. In 134 patients with single nucleotide variants (SNVs) detected in tissue biopsy, 52% of the expected SNVs were detected in cfDNA. In patients with high circulating tumour DNA (ctDNA) levels (>10% ctDNA purity as determined by lcWGS) 96% of SNVs expected from tissue sequencing were detected in cfDNA. Importantly, 40 cfDNA-unique SNVs have been detected in this cohort, highlighting the ability of cfDNA profiling to define tumour heterogeneity and identify variants missed by tissue profiling. In some cases, cfDNA-unique mutations were of clinical significance and could provide guidance for possible targeted treatments or enrolment to clinical trials.

Conclusions

Our method allows agnostic profiling of cfDNA from patients with different types of solid paediatric cancers and has the potential to replace or complement tissue biopsy testing in many clinical diagnostics situations.

Background and Aims

Circulating cell-free DNA released by tumor cells (ctDNA) provides the opportunity to minimally invasively survey clinically informative biomarkers and therapeutic targets. The INFORM registry aims to identify actionable targets on a personalized basis. Relapsed or refractory high-risk tumors, for which no further standard of care therapy is available, are characterized by next-generation sequencing (NGS) technologies. Our study aims at integrating liquid biopsies into the diagnostic INFORM pipeline and paving the way for clinical translation of these technologies.

Methods

CtDNA was derived from plasma samples and isolated according to optimized protocols. Depending on the yield of ctDNA, data availability for the primary tumor, and tumor entity, low-coverage whole-genome sequencing (lcWGS), whole-exome sequencing (WES), targeted gene panel sequencing or combinations thereof were performed. Results were compared and integrated with data obtained from standard tumor biopsies and normal control tissues following adjustment of established bioinformatics pipelines to suit liquid biopsy-derived data.

Results

The sensitivity and specificity of our approach are presented within a proof-of-concept study to compare the performance of different NGS platforms and their utility for analyzing ctDNA samples. We demonstrate robust tumor detection by lcWGS (n=115), mutation tracking by targeted approaches (n=74), and the advantages of the combinatorial use of various approaches (n=64). Despite reliable tumor monitoring, liquid biopsies aided in identifying druggable targets in metastatic disease (e.g., CDK4, CDK6, MDM2) that were absent in tumor biopsies.

Conclusions

Although limited sample volumes may impede analyses, we have demonstrated the applicability of liquid biopsies in personalized pediatric oncology. Further development of a clinical decision support system for liquid biopsies may aid in optimally realizing in-depth spatial and temporal tumor resolution.

Our study is expected to guide and accelerate the implementation of liquid biopsies in prospective pediatric clinical trials.

Background and Aims

Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC) that binds to and degrades tumor suppressor proteins (TSPs). Cjoc42 was the first small molecule inhibitor of Gankyrin developed, however IC₅₀ values > 50 μM made them unattractive for clinical use. Second generation inhibitors, also called cjoc42 derivatives, demonstrate a stronger affinity to Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives as single agents and in combination with chemotherapy.

Methods

Experiments were performed in HepG2 (HBL) and Hep3B (pediatric HCC) cell lines, which were treated with 15-256 μM of each compound. We evaluated expression of TSPs, Gankyrin, cell cycle markers, and stem cell markers by western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic assays through flow cytometry and fluorescent microscopy, synergy assays with doxorubicin and cisplatin, and methylation assays.

Results

Treatment with cjoc42 derivatives led to an increase in TSPs and dose-dependent decrease in stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1-2 in Hep3B cells. Drug synergy was seen with doxorubicin and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. A decrease in global methylation was seen after combination treatment with cisplatin in HepG2 cells, suggesting that abberant methylation may promote drug antagonism.

Conclusions

Novel therapy is needed for children with chemoresistant liver cancer. Small molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin. As newer Gankyrin inhibitors with increased cytotoxicity are developed, in vivo work will be imperative to support the use of these compounds in patients.

Background and Aims

Renal cell carcinoma (RCC) is a very rare renal tumor type in children, and evidence-based treatment guidelines are lacking. Whereas for localized pediatric RCC radical nephrectomy alone is the standard of care, advanced-stage, as well as relapsed disease, require more extensive treatment. Currently, within the SIOP-Renal Tumor Study Group (RTSG)-2016 UMBRELLA protocol, sunitinib is recommended in case of metastatic or unresectable disease. Knowledge on the effect of a broader spectrum of targeted therapies or immunotherapy is mainly based on adult RCC studies. This retrospective descriptive study focuses on the experiences of treatment with tyrosine kinase inhibitors, anti-programmed cell death 1 monoclonal antibodies or other immunotherapeutic regimens in advanced-stage and relapsed pediatric RCC patients.

Methods

This study included pediatric patients (0-18 years) with histologically diagnosed RCC, treated with targeted therapy, identified through a call to SIOP-RTSG national coordinators. All included patients were registered in SIOP- (93-01, 2001, 2016 UMBRELLA and UK-IMPORT) and AIEOP databases between 1993 and 2021. The small size and heterogeneity of this retrospective cohort allowed us only descriptive statistical analysis.

Results

Thirty-two patients were identified, with 8/32 presenting with (TNM) stage III disease, and 21/32 with stage IV disease. The majority of the patients was diagnosed with translocation type RCC (MiT-RCC) (21/32), whereas the remaining patients presented with other histological subtypes (including papillary type RCC in 5 cases and clear-cell type RCC in 2 cases). Treatment included mono- or combination therapy with a large variety of drugs, reflecting individual treatment approaches in most patients. Sunitinib alone was often administered as first choice, predominantly resulting in stable disease (47%), whereas other frequently used drugs were axitinib, cabozantinib, sorafenib and nivolumab.

Conclusions

This study provides an overview of a unique series of clinical and treatment characteristics, including disease response, of advanced-stage and relapsed pediatric RCC patients treated with targeted therapies.