Pilot Study by Liquid Biopsy in Gastrointestinal Stromal Tumors: Analysis of PDGFRA D842V Mutation and Hypermethylation of SEPT9 Presence by Digital Droplet PCR.

Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In this pilot study, we advocate for droplet digital PCR (ddPCR) to diagnose GIST in tissue samples and explore its potential for early diagnosis via liquid biopsy, focusing on the PDGFRA D842V mutation and SEPT9 hypermethylated gene. We utilized ddPCR to analyze the predominant PDGFRA mutation (D842V) in surgical tissue samples from 15 GIST patients, correlating with pathologists' diagnoses. We expanded our analysis to plasma samples to compare DNA alterations between tumor tissue and plasma, also investigating SEPT9 gene hypermethylation. We successfully detected the PDGFRA D842V mutation in GIST tissues by ddPCR. Despite various protocols to enhance mutation detection in early-stage disease, it remained challenging, likely due to the low concentration of DNA in plasma samples. Additionally, the results of Area Under the Curve (AUC) for the hypermethylated SEPT9 gene, analyzing concentration, ratio, and abundance were 0.74 (95% Confidence Interval (CI): 0.52 to 0.97), 0.77 (95% CI: 0.56 to 0.98), and 0.79 (95% CI: 0.59 to 0.99), respectively. As a rare disease, the early detection of GIST through such biomarkers is particularly crucial, offering significant potential to improve patient outcomes.

Study of the Effect of Wild-Type and Transiently Expressing CXCR4 and IL-10 Mesenchymal Stromal Cells in a Mouse Model of Peritonitis.

Sepsis due to peritonitis is a process associated with an inflammatory state. Mesenchymal stromal cells (MSCs) modulate the immune system due to the paracrine factors released and may be a therapeutic alternative. Three treatment groups were developed in a murine model of peritonitis to verify the effect of human adipose mesenchymal stem cell (hASCs). Additionally, a temporary modification was carried out on them to improve their arrival in inflamed tissues (CXCR4), as well as their anti-inflammatory activity (IL-10). The capacity to reduce systemic inflammation was studied using a local application (peritoneal injection) as a treatment route. Comparisons involving the therapeutic effect of wild-type ASCs and ASCs transiently expressing CXCR4 and IL-10 were carried out with the aim of generating an improved anti-inflammatory response for sepsis in addition to standard antibiotic treatment. However, under the experimental conditions used in these studies, no differences were found between both groups with ASCs. The peritoneal administration of hASCs or genetically modified hASCs constitutes an efficient and safe therapy in our model of mouse peritonitis.

Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations.

Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.

Analysis of Septin 9 Gene Hypermethylation as Follow-Up Biomarker of Colorectal Cancer Patients after Curative Surgery.

BACKGROUND: The Septin 9 test analyzes the methylation status of the SEPT9 gene, which appears to be hypermethylated in patients with colorectal cancer (CRC). This has been validated as a colorectal cancer screening test. Due to the high sensitivity and specificity found, the justification was to use it as a biomarker tool for monitoring minimal residual disease after radical surgery and recurrence. METHODS: A prospective study was carried out at the Fundación Jiménez Díaz University Hospital extracting peripheral blood from 28 patients and 4 healthy donors. Free circulating DNA was obtained and subsequently a PCR reaction to quantify the number of methylated genes. Samples were obtained preoperatively and postoperatively at five to seven days, one and three months after surgery. RESULTS: A total of 32 preoperative samples were analyzed. The sensitivity of the test to detect CRC was 55.6% and specificity was 100%. There were 22 postsurgical samples obtained at 5-7 days after surgery, the sensitivity to detect tumor recurrences was 100% and specificity was 75%. There were 21 samples analyzed 1 month after surgery exhibiting a sensitivity and specificity of 100% and 94.7%, respectively. At 3 months, 31 postsurgical samples were analyzed and the sensitivity and specificity were 66.7% and 80%. CONCLUSIONS: Detection of methylation of Septin 9 gene in circulating plasma DNA, obtained from a peripheral blood sample, may be a useful, non-invasive and effective method for detecting minimal residual disease and could therefore predict CRC tumor recurrences. The optimal time in our series to obtain the best prediction results based on Septin 9 methylation levels was one month after surgery. Despite these considerable findings, a study with more patients is necessary to obtain more robust conclusions.

Oncological transformation in vitro of hepatic progenitor cell lines isolated from adult mice.

Colorectal cancer cells can transfer the oncogene KRAS to distant cells, predisposing them to malignant transformation (Genometastasis Theory). This process could contribute to liver metastasis; besides, hepatic progenitor cells (HPCs) have been found to be involved in liver malignant neoplasms. The objective of this study is to determine if mouse HPCs-Oval cells (OCs)-are susceptible to incorporate Kras GAT (G12D) mutation from mouse colorectal cancer cell line CT26.WT and if OCs with the incorporated mutation behave like malignant cells. To achieve this, three lines of OCs in different conditions were exposed to CT26.WT cells through transwell co-culture for a week. The presence of KrasG12D and capacity to form tumors were analyzed in treated samples by droplet digital PCR and colony-forming assays, respectively. The results showed that the KrasG12D mutation was detected in hepatic culture conditions of undifferentiated OCs and these cells were capable of forming tumors in vitro. Therefore, OCs are susceptible to malignant transformation by horizontal transfer of DNA with KrasG12D mutation in an undifferentiated condition associated with the liver microenvironment. This study contributes to a new step in the understanding of the colorectal metastatic process.

Current and Emerging Applications of Droplet Digital PCR in Oncology: An Updated Review.

In the era of personalized medicine and targeted therapies for the management of patients with cancer, ultrasensitive detection methods for tumor genotyping, such as next-generation sequencing or droplet digital polymerase chain reaction (ddPCR), play a significant role. In the search for less invasive strategies for diagnosis, prognosis and disease monitoring, the number of publications regarding liquid biopsy approaches using ddPCR has increased substantially in recent years. There is a long list of malignancies in which ddPCR provides a reliable and accurate tool for detection of nucleic acid-based markers derived from cell-free DNA, cell-free RNA, circulating tumor cells, extracellular vesicles or exosomes when isolated from whole blood, plasma and serum, helping to anticipate tumor relapse or unveil intratumor heterogeneity and clonal evolution in response to treatment. This updated review describes recent developments in ddPCR platforms and provides a general overview about the major applications of liquid biopsy in blood, including its utility for molecular response and minimal residual disease monitoring in hematological malignancies or the therapeutic management of patients with colorectal or lung cancer, particularly for the selection and monitoring of treatment with tyrosine kinase inhibitors. Although plasma is the main source of genetic material for tumor genomic profiling, liquid biopsy by ddPCR is being investigated in a wide variety of biologic fluids, such as cerebrospinal fluid, urine, stool, ocular fluids, sputum, saliva, bronchoalveolar lavage, pleural effusion, mucin, peritoneal fluid, fine needle aspirate, bile or pancreatic juice. The present review focuses on these "alternative" sources of genetic material and their analysis by ddPCR in different kinds of cancers.