The clinical management of cancer has evolved in recent years towards more personalized strategies that require a comprehensive knowledge of the complex molecular features involved in tumor growth and evolution, and the development of drug resistance mechanisms leading to disease progression. Droplet digital PCR (ddPCR) has become one of the most accurate and reliable tools for the examination of genetic alterations in a wide variety of cancers because of its high sensitivity and specificity. ddPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples. This methodology has proven useful for the evaluation of archival tumor tissues, where poor DNA quality and limited sample availability are major obstacles for standard methods, providing less subjective and more automated quantitative results. However, most applications of ddPCR in cancer are focused on liquid biopsies (including cell-free DNA as well as circulating tumor cells) because these represent non-invasive alternatives to tissue biopsies that can more accurately reflect intratumoral heterogeneity and track the dynamic changes in tumor burden that occur in response to treatment at different times during follow-up. A broad spectrum of molecular markers have been interrogated in blood using ddPCR for diagnostic, predictive, and monitoring purposes in various malignancies. Emerging alternative approaches using other body fluids such as cerebrospinal fluid and urine are also currently being developed. This article aims to give a complete overview of ddPCR applications for molecular screening in oncology.

SUMMARY Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels.

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Our aim was to characterize and validate that the location and age of onset of the tumor are both important criteria to classify colorectal cancer (CRC). We analyzed clinical and molecular characteristics of early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and we compared each tumor location between both ages-of-onset. In right-sided colon tumors, early-onset cases showed extensive Lynch syndrome (LS) features, with a relatively low frequency of chromosomal instability (CIN), but a high CpG island methylation phenotype. Nevertheless, late-onset cases showed predominantly sporadic features and microsatellite instability cases due to BRAF mutations. In left colon cancers, the most reliable clinical features were the tendency to develop polyps as well as multiple primary CRC associated with the late-onset subset. Apart from the higher degree of CIN in left-sided early-onset cancers, differential copy number alterations were also observed. Differences among rectal cancers showed that early-onset rectal cancers were diagnosed at later stages, had less association with polyps, and more than half of them were associated with a familial LS component. Stratifying CRC according to both location and age-of-onset criteria is meaningful, not only because it correlates the resulting categories with certain molecular bases, but with the confirmation across larger studies, new therapeutical algorithms could be defined according to this subclassification.

The usefulness of local collagenase in therapeutic approaches to solid tumors has been tested recently. In this study, we evaluate the safety and efficacy of intraperitoneal collagenase associated or not to mitomycin for treatment of colorectal peritoneal metastases in an experimental rat model. Using a fixed-dose procedure, we found that a dose of collagenase of 37 IU/mL administered for 15 min with a hyperthermia pump at 37.5 °C, both in isolation or associated to sequential treatment with intraperitoneal mitomycin, led to a macroscopic decrease in tumor volume as evaluated by the modified peritoneal cancer index (mPCI). Concerning the safety of the procedure, the animals showed no physiological or behavioral disorders during 8 weeks of follow-up. Local treatment for peritoneal metastases of colorectal origin with intraperitoneal collagenase has proved safe and effective in an experimental murine model. Therefore, the stroma-first approach by enzymatic breakdown of collagen from the tumor's extracellular matrix provides a new therapeutic target for colorectal peritoneal metastases.