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Liquid biopsies enable noninvasive therapy monitoring in patients with solid tumors. Specific serum markers such as proteins, hormones, or enzymes released from tumor cells or in response to tumor growth can be used for quantification of the tumor burden. However, only a fraction of pediatric tumors has none of these serum markers, but tumor-specific genetic alterations represent reliable alternatives. Here we describe a method for using genomic fusion sequences as liquid biopsy markers in Ewing sarcoma patients.

Droplet digital PCR (ddPCR) allows for accurate quantification of genetic events such as copy number variation and single nucleotide variants. Probe-based assays represent the current "gold-standard" for detection and quantification of these genetic events. Here, we introduce a cost-effective single color ddPCR assay that allows for single genome resolution quantification of copy number and single nucleotide variation.

Liquid biopsy of cancers is an area of increasing interest in medical practice for the surveillance, management, and potential detection of malignant cells, using minimally invasive collection of body fluids. A liquid biopsy is particularly useful for metastatic cancers, which may be difficult to be sampled by core biopsy, due to difficulty of access or an occult location. Access to DNA shed from esophageal adenocarcinoma can enable the detection of mutations confirming the presence of malignant cells or the evolution of clonal lines with altered treatment response profiles. In this chapter, we detail a method for the isolation of cell-free DNA from blood plasma and DNA associated with exosomes in blood from patients with esophageal adenocarcinoma.

Digital PCR (dPCR) is a highly accurate method to determine DNA concentration. In dPCR, DNA is portioned into many discrete single entities, and these are analyzed individually for the presence or absence of a target molecule of interest. Here we describe how digital PCR can be employed to determine the presence of oncogenic amplification through noninvasive analysis of circulating free DNA (cfDNA), and exemplify this approach by developing a plasma circulating free DNA dPCR assay for HER2 copy number.

Breast cancer is the leading cause of cancer in women and the second leading cause of cancer-related death. There are many subtypes of breast cancer, which can be identified through the process of molecular and genetic profiling. While the current standard of care utilizes tumor tissue biopsy to subclassify breast cancer, plasma tumor DNA (ptDNA) can be detected through droplet digital PCR (ddPCR) of plasma obtained from a simple blood draw. Tissue biopsy is not only more invasive but because tumors exhibit heterogeneity it can be less accurate. Blood collects DNA shed from normal and cancerous cells alike, thus ddPCR of plasma offers a broader picture of a cancer's genetic makeup. This chapter summarizes how patients with breast cancer can be screened for specific cancerous mutations in both tissue and plasma through the use of ddPCR.

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.

Digital PCR techniques are ideally suited for accurately quantifying trace amounts of target DNA sequences, such as tumor-derived mutant DNA that is present in the blood circulation of patients with cancer. Here, we describe an approach marrying low-coverage whole-genome sequencing of tumor tissues, to enumerate chromosomal rearrangement breakpoints, together with droplet digital PCR (ddPCR)-based personalized rearrangement assays to cost-effectively monitor circulating tumor DNA levels at multiple time-points during the clinical course. The method is generally applicable to essentially any cancer patient, as all cancers harbor unstable genomes, and may have uses for measuring minimal residual disease, response to therapy, and early detection of metastasis.