Neuroblastoma (NB) is the most common and deadly solid tumour in children. Despite the development of new treatment options for high-risk NB, over half of patients relapse and five-year survival remains at 40–50%. Therefore, novel treatment strategies aimed at providing long-term disease remission are urgently sought. ALK, encoding the anaplastic lymphoma kinase receptor, is altered by gain-of-function point mutations in around 14% of high-risk NB and represents an ideal therapeutic target given its low or absent expression in healthy tissue postnatally. Small-molecule inhibitors of Anaplastic Lymphoma Kinase (ALK) approved in ALK fusion-positive lung cancer are currently undergoing clinical assessment in patients with ALK-mutant NB. Parallel pre-clinical studies are demonstrating the efficacy of ALK inhibitors against common ALK variants in NB; however, a complex picture of therapeutic resistance is emerging. It is anticipated that long-term use of these compounds will require combinatorial targeting of pathways downstream of ALK, functionally-related ‘bypass’ mechanisms and concomitant oncogenic pathways.

Circulating-free DNA (cfDNA) is under investigation as a liquid biopsy of cancer for early detection, monitoring disease progression and therapeutic response. This systematic review of the primary cfDNA literature aims to identify and evaluate factors that influence recovery of cfDNA, and to outline evidence-based recommendations for standardization of methods. A search of the Ovid and Cochrane databases was undertaken in May 2018 to obtain relevant literature on cfDNA isolation and quantification. Retrieved titles and abstracts were reviewed by two authors. The factors evaluated include choice of specimen type (plasma or serum); time-to-processing of whole blood; blood specimen tube; centrifugation protocol (speed, time, temperature and number of spins); and methods of cfDNA isolation and quantification. Of 4,172 articles identified through the database search, 52 proceeded to full-text review and 37 met the criteria for inclusion. A quantitative analysis was not possible, due to significant heterogeneity in methodological approaches between studies. Therefore, included data was tabulated and a textual qualitative synthesis approach was taken. This is the first systematic review of methodological factors that influence recovery and quantification of cfDNA, enabling recommendations to be made that will support standardization of methodological approaches towards development of blood-based cancer tests.

Improving early detection of colorectal cancer (CRC) is a key public health priority as adenomas and stage I cancer can be treated with minimally invasive procedures. Population screening strategies based on detection of occult blood in the feces have contributed to enhance detection rates of localized disease, but new approaches based on genetic analyses able to increase specificity and sensitivity could provide additional advantages compared to current screening methodologies. Recently, circulating cell-free DNA (cfDNA) has received much attention as a cancer biomarker for its ability to monitor the progression of advanced disease, predict tumor recurrence and reflect the complex genetic heterogeneity of cancers. Here, we tested whether analysis of cfDNA is a viable tool to enhance detection of colon adenomas. To address this, we assessed a cohort of patients with adenomas and healthy controls using droplet digital PCR (ddPCR) and mutation-specific assays targeted to trunk mutations. Additionally, we performed multiregional, targeted next-generation sequencing (NGS) of adenomas and unmasked extensive heterogeneity, affecting known drivers such as APC, KRAS and mismatch repair (MMR) genes. However, tumor-related mutations were undetectable in patients’ plasma. Finally, we employed a preclinical mouse model of Apc-driven intestinal adenomas and confirmed the inability to identify tumor-related alterations via cfDNA, despite the enhanced disease burden displayed by this experimental cancer model. Therefore, we conclude that benign colon lesions display extensive genetic heterogeneity, that they are not prone to release DNA into the circulation and are unlikely to be reliably detected with liquid biopsies, at least with the current technologies.

Molecular analysis of nucleic acid and protein biomarkers is becoming increasingly common in paediatric oncology for diagnosis, risk stratification and molecularly targeted therapeutics. However, many current and emerging biomarkers are based on analysis of tumour tissue, which is obtained through invasive surgical procedures and in some cases may not be accessible. Over the past decade, there has been growing interest in the utility of circulating biomarkers such as cell-free nucleic acids, circulating tumour cells and extracellular vesicles as a so-called liquid biopsy of cancer. Here, we review the potential of emerging circulating biomarkers in the management of neuroblastoma and highlight challenges to their implementation in the clinic.

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.

Lung cancer is the leading cause of cancer-related death. Two-thirds of cases are diagnosed at an advanced stage that is refractory to curative treatment. Therefore, strategies for the early detection of lung cancer are urgently sought. Total circulating free DNA (cfDNA) and tumour-derived circulating tumour DNA (ctDNA) are emerging as important biomarkers within a ‘liquid biopsy’ for monitoring human disease progression and response to therapy. Owing to the late clinical diagnosis of lung adenocarcinoma, the potential for cfDNA and ctDNA as early detection biomarkers remains unexplored. Here, using a Cre-regulated genetically engineered mouse model of lung adenocarcinoma development, driven by Kras^G12D (the Kras^LSL-G12D mouse), we serially tracked the release of cfDNA/ctDNA and compared this with tumour burden as determined by micro-computed tomography (CT). To monitor ctDNA, a droplet digital PCR assay was developed to permit discrimination of the Kras^Lox-G12D allele from the Kras^LSL-G12D and Kras^WT alleles. We show that micro-CT correlates with endpoint histology and is able to detect pre-malignant tumours with a combined volume larger than 7 mm³. Changes in cfDNA/ctDNA levels correlate with micro-CT measurements in longitudinal sampling and are able to monitor the emergence of lesions before the adenoma-adenocarcinoma transition. Potentially, this work has implications for the early detection of human lung adenocarcinoma using ctDNA/cfDNA profiling. A video abstract for this article is available at https://youtu.be/Ku8xJJyGs3U. This article has an associated First Person interview with the joint first authors of the paper.