Biomarkers for Prostate Cancer – Beyond the PSA test

Biomarkers can be DNA, RNA, proteins, or chemicals produced by the body that indicate the presence or severity of a disease. Biomarkers can be used as signals for early detection, to help make treatment choices, or to monitor disease progression or response to certain treatments. Biomarkers can be detected in any tissue or fluid and are a critical component of a personalised medicine approach to cancer treatment.

The Need for New Biomarkers

The PSA test

The Prostate Specific Antigen (PSA) is a protein produced by the prostate gland. Elevated PSA levels often indicate prostate cancer, making it a key biomarker for diagnosis. The PSA test involves drawing blood to measure PSA levels, but it has limitations. Factors like age, ethnicity, urinary tract infections, and recent ejaculation can affect PSA levels, complicating interpretation. Moreover, PSA testing may detect tumours that are unlikely to cause harm, leading to unnecessary treatment in anywhere from 2% to 67% of cases. This underscores the need for novel biomarkers to improve prostate cancer detection and treatment decisions.

A New Alternative – Liquid Biopsies

Molecular profiling of tumours complements the PSA test by providing a definitive diagnosis through tissue biopsy and histological analysis. This approach supports decisions made on personalised cancer treatment, predicts patient responses, detects drug resistance, and monitors tumour relapse. In managing metastatic prostate cancer, androgen deprivation therapy (ADT) is pivotal, but resistance to main ADT drugs like abiraterone and enzalutamide can occur if ARV7 is detected in cancer cells, driving demands to conduct new ADT drug trials. Despite precious insights from tissue biopsies, they are invasive and offer only a snapshot of information. Liquid biopsies present a promising alternative, enabling easier and repeatable sampling primarily from blood, with ongoing research exploring urine, bile, saliva, and cerebrospinal fluid as potential resources for biomarkers like DNA, RNA, and proteins encapsulated in either vesicles or within circulating tumor cells. 

Importance of the Pre-Analytical Workflow

Successful cancer biomarker detection through liquid biopsies relies heavily on the pre-analytical workflow, which includes analytes preservation at the sampling site, meticulous handling during transportation, and thorough sample processing. 

Blood Collection and Preservation

Blood drawn for liquid biopsies often cannot be processed immediately on-site, making it crucial to preserve and stabilize analytes during transportation. Methods like Norgen tubes (Cat. 63950) stabilize nucleated cells osmotically, minimize leukocyte lysis and preserve biomarker integrity. Norgen tubes have been commonly chosen in several studies for their high-quality plasma yields with minimal hemolysis, enabling parallel extraction of cfDNA and cfRNA. This approach supports sensitive detection of tumour-specific alterations using techniques like droplet digital PCR and low-coverage whole-genome sequencing.

Urine Collection and Preservation

Urine is gaining attention in the liquid biopsy field as a non-invasive alternative to blood/plasma sampling, offering easy access, high patient compliance, and a reflection of overall health through kidney filtration. However, challenges like microbial contamination and increased nucleases can affect analysis if urine is not properly preserved. Norgen’s Urine Preservation tubes (Cat. 18113) have been used to collect samples from 43 patients with bladder cancer and healthy donors, extracting cell-free DNA for genetic mutation detection across 118 cancers. This method showed 83.7% sensitivity and 100% specificity for bladder cancer detection. While this study focused on urological cancers, advancements in ultra-short cfDNA extraction methods could extend liquid biopsy applications to non-urological cancers. In another approach, researchers used Norgen devices for LC/MS-based untargeted metabolomics, identifying prostate cancer-specific metabolites in urine samples.

Nucleic Acid Extraction

Cell-free DNA/RNA

Cell-free DNAs (cfDNA) are fragmented DNA released mainly from dying or cancerous cells. Healthy individuals primarily release cfDNA from apoptotic cells, typically 185-200 base pairs long. In cancer patients, cfDNA levels rise with more varied fragment lengths. Recent studies on cfDNA as a prostate cancer (PCa) biomarker shows high specificity but limited sensitivity. Further research into cfDNA’s epigenetic and fragmentomic aspects holds promise for improving PCa diagnosis.

MicroRNAs (miRNAs) in body fluids have low concentrations, limiting their use as cancer biomarkers. To enhance their detection, ultrasound treatment was found to increase the release of miRNAs from both androgen-dependent (AD) and -independent (AI) PCa cells. This method identified four PCa-related miRNAs whose levels rose after ultrasound treatment and were higher in PCa patients than controls. Ultrasound shows potential for discovering new cell-free miRNA biomarkers for cancer diagnosis and prediction.7

Exosomal DNA/RNA

Exosomes are small extracellular vesicles (40-150 nm) released by all cell types, including tumor cells. They are stable in body fluids due to their lipid bilayer membrane and carry DNA, RNA, and proteins. Exosomes play a crucial role in intercellular communication, capable of targeting specific cells by fusing with their membranes and releasing their contents. This cargo often influences tumor progression, as tumors and diseased cells release more exosomes compared to healthy cells. 

Researchers have studied exosomal serum miRNAs (ex-miRNA) in individuals ranging from healthy controls to those with benign prostatic hyperplasia (BPH) and aggressive prostate cancer. Using Nanostring nCounter, they identified differentially expressed miRNAs, including miR-1246, a tumor-suppressing miRNA found to be downregulated in clinical prostate cancer tissues and cell lines. This suggests that miR-1246 holds promise as a biomarker for prostate cancer diagnosis and predicting disease aggressiveness.

Biomarker Detection Methods

Genomics

Certain inherited, or germline, variants predispose individuals to higher cancer risks, serving as valuable biomarkers for cancer risk assessment. Rare variants with high penetrance, like BRCA1 and BRCA2 mutations, significantly increase the risk of breast and ovarian cancers. In men, these mutations also elevate the risk of aggressive prostate cancer.

Somatic variations are another critical category of cancer biomarkers. Cancer cells exhibit genomic instability, accumulating somatic mutations as they adapt to their microenvironment. These mutations range from structural abnormalities (e.g., translocations, copy number alterations) to single base pair changes. They can be detected in tumour tissue and in circulating tumor DNA (ctDNA), released from cancer cells through lysis or active secretion.

Epigenomics

Epigenetic variations, such as DNA methylation and histone modifications, alter gene and protein expression without changing the DNA sequence itself, making them valuable cancer biomarkers. DNA methylation, in particular, is significant as many tumors exhibit reduced DNA methylation, linked to genomic instability and damage.

Detecting DNA methylation biomarkers in liquid biopsies offers advantages over mutation detection, with higher sensitivity and specificity for early cancer stages and residual disease. These changes are early indicators and specific to the tissue affected. Chen et al. developed the “PanSeer” plasma DNA methylation panel, testing it on over 120,000 individuals without cancer symptoms. They followed them for up to 4 years and found that PanSeer detected stomach, esophageal, colorectal, lung, and liver cancers in 95% of asymptomatic individuals up to 4 years before diagnosis.

Fragmentomics

Fragmentomics is a new approach in liquid biopsy research that analyzes the fragmentation patterns of cell-free DNA (cfDNA) across the entire population, rather than specific mutations. These patterns, influenced by epigenetic regulation, provide insights into cancer detection and tissue origin. Key properties examined include fragment length distribution, end-motif sequences, and nucleosome footprint.

Researchers recently used patient-derived organoids from normal and cancerous tissues to extract cfDNA and analyzed it using fragmentomic analysis. They found distinct patterns between tissues in different states (proliferative vs. apoptotic), highlighting the utility of organoid models in studying cfDNA biology.

In prostate cancer, Helzer et al. applied fragmentomic analysis to cfDNA from plasma, focusing on coding regions with targeted panels to reduce sequencing costs. Using machine learning models, they successfully distinguished cancer from non-cancer patients and identified specific tumor types and subtypes, showing promise for prostate cancer screening.

Related Norgen Products

Norgen Biotek has an entire Liquid Biopsy workflow with products for all of the steps including sample preservation and nucleic acid isolation, and Norgen even offers sequencing services. Norgen offers preservation devices for both Blood and Urine. Norgen also has kits for purifying cell-free DNA from plasma and cell-free RNA from plasma/serum or exosomes. Norgen offers kits to isolate exosomes from Plasma/Serum, as well as from Urine. Exosomal RNA purification kits, Plasma-specific exosomal RNA purification kits, Urine-specific exosomal RNA purification kits, and Cell-free RNA purification kits. To help with sequencing based analyses Norgen provides Small RNA Library Preparation kits and also offers complete Sequencing services.

References

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