Mutations in tumor-suppressor genes are associated with many types of cancers. In addition, epigenetic changes (such as DNA methylation) of tumor-suppressor genes are also associated with tumorigenesis (Otani et al., 2013. Expert Rev Mol Diagn \(13: 445-455\) ). (a) How might hypermethylation of the \(p 53\) gene promoter influence tumorigenesis? (b) Knowing that tumors release free DNA into certain surrounding body fluids through necrosis and apoptosis (Kloten et al., 2013. Breast Cancer Res. \(15(1): \mathrm{R} 4\) ), outline an experimental protocol for using human blood as a biomarker for cancer and as a method for monitoring the progression of cancer in an individual.

Hypermethylation occurs when there is an increased amount of methylation within a gene promoter region. This can lead to the silencing of tumor-suppressor genes, such as p53. When the p53 gene promoter is hypermethylated, the gene is less likely to be transcribed and translated, leading to a reduction in the production of the p53 protein. The p53 protein plays a crucial role in preventing tumor formation by repairing damaged DNA and triggering cell death when repair is not possible. Therefore, hypermethylation of the p53 gene promoter could contribute to tumorigenesis by suppressing the production of the p53 protein, thus reducing its function as a tumor suppressor.

Step 1: Sample collection Collect blood samples from patients with cancer and healthy individuals (control group) at appropriate time points. This can be done using standard phlebotomy techniques. Step 2: Isolate cell-free DNA (cfDNA) Isolate cfDNA from the blood samples using appropriate methods, such as column-based or magnetic bead-based extraction kits specifically designed for cfDNA isolation. Step 3: Quantify cfDNA Measure the concentration of cfDNA in each sample using fluorometric methods or quantitative polymerase chain reaction (qPCR). Step 4: Analyze for specific tumor markers in cfDNA Perform polymerase chain reaction (PCR) or next-generation sequencing (NGS) to analyze the presence of tumor-specific markers, such as specific mutations or methylation patterns, in the cfDNA samples. Step 5: Compare and analyze results Compare the levels of cfDNA and tumor-specific markers between cancer patients and the control group. Higher levels of cfDNA and altered tumor-specific markers in cancer patients could indicate the presence and progression of cancer. Step 6: Monitor cancer progression Repeat the analysis at different time points during the course of cancer treatment to monitor the effectiveness of the treatment and disease progression. Decreased levels of cfDNA and normalized tumor-specific markers could suggest a positive response to treatment. Step 7: Analyze results and draw conclusions Analyze the data to determine whether blood samples can serve as a reliable biomarker for cancer detection and monitoring and draw conclusions based on the results.