Neuroblastoma is one of the most common cancers in children, and its incidence has increased by 7% every 10 years between 1985 and 2015 [22, 24]. Advances have been made in the diagnosis and therapy of NB, including better radiological imaging, cytological, biochemical, and molecular techniques; however, the 5-year survival rate of patients with high-risk NB remains below 50% [11, 25, 26]. Many factors contribute to this disappointing outcome, most notably the persistence of chemoresistant minimal residual disease (MRD), which is responsible for disease recurrence in >50% of patients with high-risk NB [1, 14, 24, 27]. Therefore, accurate detection of MRD is crucial to enable prompt therapeutic action.
Finding biomarkers to evaluate prognosis or response to treatment is an intense area of cancer research. Plasma cfDNA has been extensively investigated as a potential biomarker, especially for malignant metastatic cancers [28]. Compared with tissue-based histological or imaging tests, which are sensitive to sampling bias and poor repeatability, measurement of plasma cfDNA represents a minimally invasive method to monitor tumor burden and thus act as both a clinical and pathological biomarker [29]. cfDNA levels have been shown to be significantly higher in patients with cancer compared with benign disease [30], and is a potential marker of the therapeutic response and prognosis of patients with a wide range of cancers, such as lung cancers and gastrointestinal malignancies [17, 31-33]. cfDNA is thought to originate predominantly from tumor cells and hematopoietic cells [34]. We previously showed that inflammation, transfusion, and therapy with granulocyte-colony stimulating factor are key clinical factors affecting the quantification of cfDNA [35]. To avoid detection of cfDNA from non-tumor cells, blood should not be sampled for tumor cfDNA analysis in these three settings.
Quantification of cfDNA by qPCR has three main advantages over digital PCR and next generation sequencing for detection tumor burden; namely, ease of performance, common use, and low cost. However, its disadvantages include lower sensitivity and detection of a limited number of genomic loci per analysis. Several other methods have been proposed to improve the sensitivity of detection of tumor-derived plasma cfDNA, including gene-specific panel profiling, whole exome/genome sequencing, and digital PCR [36, 37].
Current digital PCR techniques have high sensitivity to detect low allele fractions variants [38]. Whole exome/genome sequencing offers a comprehensive analysis of tumor mutations and has broad applications, but it is an expensive method [39]. Thus, quantification of plasma cfDNA by qPCR represents a relatively simple, inexpensive, and reproducible method to monitor tumor burden.
We previously demonstrated that plasma cfDNA levels correlated strongly with tumor burden in children with NB [23], and could potentially serve as a more effective biomarker than LDH, which is widely used in the clinic. Furthermore, plasma cfDNA concentrations were significantly lower in patients with PR compared with SD, and the concentrations were dynamically associated with changing tumor burden in response to chemotherapy [35]. However, whether cfDNA could serve as an effective molecular marker for recurrence was unknown. Here, we showed that plasma cfDNA levels increased significantly before the diagnosis of recurrence; however, this did not occur in all patients with recurrence, which could be due to a number of factors, including tumor stage, tumor heterogeneity, and other clinicopathological characteristics [19]. The clinical features associated with disease recurrence in NB are complex, and include the metastatic site, tumor cell abundance, and tumor aggressiveness. Therefore, it is not surprising that cfDNA levels vary among individuals with recurrent NB.
Quantification of both cfDNA in plasma and tumor cell-derived mRNA in peripheral blood may be useful for detecting MRD in NB patients [40-42]. In patients with high-risk NB, qPCR-mediated detection of tyrosine hydroxylase (TH) and paired-like homeobox 2B (PHOX2B) mRNA levels is a sensitive and specific method for detecting MRD [41, 42]. However, whether plasma cfDNA or TH/PHOX2B mRNA is the superior marker is difficult to determine. The heterogeneity of NB might suggest that a threshold value of both cfDNA and TH/PHOX2B mRNA should be exceeded to declare MRD positivity. Notably, circulating cfDNA may be more stable than mRNA [43], and fewer steps are required for the quantification of cfDNA compared with mRNA, making it less expensive. We are currently investigating the utility of PHOX2B mRNA monitoring in NB patients and whether a correlation exists between PHOX2B mRNA and cfDNA levels.
In conclusion, we have shown here that a significant rise in plasma cfDNA concentration occurs between 1 and 3 months before disease recurrence in patients with high-risk NB. Thus, plasma cfDNA could be a promising marker of imminent disease recurrence, or at least a useful monitoring tool, during maintenance treatment for this patient population.