Question

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 \(T P 53\) gene promoter influence tumorigenesis? (b) Knowing that tumors release free DNA into certain surrounding body fluids through necrosis and apoptosis Kloten et al. \([(2013) . \text { Breast Cancer Res.} 15(1): \mathrm{R} 4]\) 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.

Short answer

Answer: Hypermethylation of the TP53 gene promoter can lead to the silencing of the TP53 tumor suppressor gene, which is responsible for preventing tumor formation. This silencing can increase the risk of tumorigenesis. Human blood can be used as a biomarker for monitoring cancer progression by isolating circulating free DNA (cfDNA), quantifying the amount of cfDNA, detecting tumor-specific mutations and methylation markers, and correlating the levels of cfDNA or biomarkers with clinical data. This process is repeated regularly to monitor cancer progression and adjust treatment strategies based on the results.

Step-by-step solution

(a) Effect of hypermethylation on TP53 gene promoter and tumorigenesis

Hypermethylation of the TP53 gene promoter can lead to the silencing of the TP53 tumor suppressor gene. TP53 is a crucial gene in preventing tumor formation, as it is responsible for stopping cell division when DNA damage is detected and facilitating DNA repair or apoptosis if the damage is irreparable. When the TP53 gene is silenced due to hypermethylation, it loses its ability to prevent tumor formation, and this can lead to an increased risk of tumorigenesis.

(b) Experimental protocol for using human blood as a biomarker for cancer monitoring

Here is a step-by-step experimental protocol for using human blood as a biomarker for cancer and monitoring the progression of cancer in an individual: Step 1: Blood sample collection Collect blood samples from the individual at regular intervals, such as every three months, to monitor the progression of cancer over time. Step 2: Isolation of circulating free DNA (cfDNA) Isolate cfDNA from the collected blood samples using a nucleic acid extraction kit or other standard techniques. Step 3: Quantification of cfDNA Quantify the amount of cfDNA in each sample using methods such as quantitative PCR (qPCR) or digital droplet PCR (ddPCR). By comparing the concentration of cfDNA in successive blood samples, one can assess if the quantity of cfDNA is increasing (which may indicate tumor progression) or decreasing (which may indicate tumor regression). Step 4: Detection of tumor-specific mutations and methylation markers Analyze the cfDNA for specific gene mutations or epigenetic changes such as methylation markers that are associated with the patient's cancer. This can be done using targeted next-generation sequencing (NGS), methylation-specific PCR, or other molecular techniques. Step 5: Correlate cfDNA or biomarker levels with cancer progression Correlate the amount of cfDNA or presence of tumor-specific mutations or methylation markers with the patient's clinical data (e.g., tumor size, metastasis, response to therapy) to determine if the levels of cfDNA or specific biomarkers can be used to monitor cancer progression. Step 6: Continuous monitoring Repeat the entire protocol regularly (e.g., every three months) to monitor the individual's cancer progression over time and adjust treatment strategies based on the results.

Key concepts

Hypermethylation of the TP53 Gene

The gene TP53 is like a guardian in our cells, ensuring DNA is accurately copied during cell division and preventing damaged cells from turning cancerous. But when the TP53 gene is hypermethylated, it's like putting a veil over its eyes; the gene becomes silent and unable to perform its protective role. This silenced state hampers its ability to stop a cell with damaged DNA from dividing, thus increasing the risk of cancer development.
Hypermethylation as a Foe: Normally, methylation is a natural occurring process affecting gene activity. However, in the case of TP53, too much methylation, or hypermethylation, on its promoter region (a gene's 'on' switch) can suppress its activity. Unfortunately, when TP53 can't work properly, cells with genetic damage may proliferate, leading to tumor growth.
Understanding the Methylation Markers: Knowing the hypermethylation status of TP53 could serve as a clue to a person’s cancer risk or the presence of hidden tumors. Therefore, monitoring the methylation levels of TP53 provides critical information to healthcare professionals.

Cancer Biomarkers

Cancer biomarkers are like fingerprints; they help identify the presence of cancer in the body and provide insights into the disease's nature. These biomarkers can be various substances, including proteins, DNA mutations, or certain changes in gene expression profiles that are associated with cancer growth.
Role of Biomarkers: They are incredibly useful in early detection, diagnosis, treatment planning, and monitoring of cancer. For example, elevated levels of prostate-specific antigen (PSA) in the blood can indicate prostate cancer, while changes in the BRCA genes might suggest a higher risk for breast and ovarian cancers.
Biomarkers in Action: By keeping an eye on specific biomarkers, doctors can not only spot cancer but also track how well a patient responds to treatment, making these biomarkers valuable allies in the fight against cancer.

Circulating Free DNA

Circulating free DNA (cfDNA) is like tiny messengers in our bloodstream, carrying information about our genetic makeup. When cells in our body die, they release fragments of DNA into the bloodstream, including healthy cells and, potentially, cancerous cells.
cfDNA as a Cancer Indicator: The amount of cfDNA can increase in cancer patients, which makes it a valuable tool for studying tumors non-invasively. By analyzing cfDNA, scientists can gather information about specific mutations or other changes that cancer may be causing in the DNA.
Advancement in Detection: Thanks to technological advancements, we can now extract and examine cfDNA for abnormalities, providing a 'liquid biopsy' that offers a glimpse into the state of a person's cancer without the need for more invasive traditional biopsies.

Cancer Monitoring

Monitoring cancer is like tracking the path of a storm; it demands constant vigilance to predict its trajectory and impact. For patients undergoing cancer therapy, regularly tracking the disease’s progress is crucial to adjusting treatment strategies accordingly.
Tools for Tracking: Through blood tests, medical imaging, and biomarkers, doctors can observe tumor size, spread, and response to treatments. This ongoing surveillance helps in deciding whether to continue the current course of action or to try new methods.
Patient-Specific Monitoring: Since each patient’s cancer journey is distinct, personalized monitoring plans are paramount. Testing for cell-free DNA and other biomarkers in the blood provides specific details on individual tumors, allowing for tailored treatments.

Epigenetic Changes in Cancer

Epigenetic changes are like subtle switches that control how our genes are expressed. Unlike genetic mutations that alter the DNA sequence, epigenetic changes can turn genes on or off without changing their sequence.
Cancer - An Epigenetic Puzzle: In cancer, these epigenetic modifications can be just as harmful as genetic mutations because they can silence tumor-suppressor genes or activate oncogenes. Examples of these changes include methylation, histone modification, and microRNA regulation.
Relevance of Epigenetics: Epigenetic changes are central to cancer development and progression. Understanding these modifications opens doors for targeting them therapeutically to 'reset' the expression of genes involved in cancer, offering a promising area for new treatments.