Liquid biopsy in early detection and monitoring of cancer

The ability to detect cancer at earlier stages has the potential to reduce cancer mortality. Currently, tissue biopsy is the classic method for cancer diagnosis in solid tumors but its application is limited in early, non-symptomatic cancers. Utilizing repeated conventional tissue biopsies to detect tumor progression is an invasive, painful and impractical approach that may lead to bleeding, cancer cell seeding and it is also not able to detect intra-tumorheterogeneity. The development of liquid biopsy techniques overcomes all the addressed limitations of tissue biopsy and is less expensive compared with surgical tissue biopsy. Currently, liquid biopsies are largely utilized to gather information on tumors in order to detect early stage cancer, select personalized medicine and prognosis along with diagnosis of cancer. Liquid biopsy has the potential for screening and real-time monitoring of disease progression. Early detection of it plays a crucial role in the prevention and treatment of cancer. It is a less-invasive screening tool for solid tumor and able to diagnose typical advanced stages of cancers which are not diagnosed until tap advance stage, like lung and pancreatic carcinoma.

Mechanism: How liquid biopsy works!

There are several mechanisms by which liquid biopsies can be utilized in early detection and monitoring the progression of various types of cancer. The primary analytes of liquid biopsy which are exhaustively explored for early detection and monitoring of cancer are cell free DNA (cfDNA), cell tumor DNA (ctDNA) and circulatory tumor cells (CTCs) with established methods for detection of them from plasma.

1. 1. Cell free DNA (cfDNA)

The cfDNA released into the bloodstream due to apoptosis, necrosis and cell disruption. The cfDNA released in cancer patients is 50 times more than that of healthy individuals. Because of release of it in healthy individuals, it cannot be exclusively linked to tumor cells. The cfDNA released into the bloodstream from cancerous tumor cells has been termed as ctDNA. The size of ctDNA molecules is shorter than cfDNA, which may also be useful in the isolation of ctDNA from healthy cfDNA. The half-life of cfDNA and ctDNA molecules in circulation are less than three hours due to which isolation of them are considered as monitoring the severity of the disease in real time.

The application of cfDNA in cancer early detection has been much improved in recent years. Current studies have demonstrated the potential use of 5hmC signatures in cfDNA as biomarkers for predicting the tumor type and stages of cancer. The 5hmC biomarker from cfDNA was found highly predictive in colorectal and gastric cancers compared to conventional biomarkers in plasma. The 5hmC biomarker was found as a robust biomarker of plasma cfDNA for early diagnosis of hepatocellular carcinoma. However, detection of 5hmC requires highly sensitive methods due to lower concentration in blood cells.

The advanced type of PCR and NGS methods can help to identify and quantify less than 0.01% of allelic mutations in cfDNA. The simultaneous gene sequencing of ctDNA in cerebrospinal fluid (CSF) and cfDNA in plasma, indicated that they have the ability to diagnose early stage glioma. Studies also reported that methylated cfDNA immunoprecipitation and high throughput sequencing are a highly sensitive assay for the detection of early stage tumors. Moreover, plasma and urine cfDNA were used to distinguish patients with renal cell carcinoma from healthy people.

It is reported that cfDNA is more sensitive and specific than other biomarkers for early detection and monitoring of cancer progression.

1. 2. Circulating tumor cells (CTCs)

Circulating tumor cells are released into the bloodstream from tumors. Proper quantification of CTCs which originate from both sites, primary tumor and metastatic sites make them also a useful biomarker for diagnosis in early stage, staging, monitoring and prognosis of cancer.

Compared with CTCs, cfDNA has higher sensitivity and specificity in early detection of cancer. The transfer of CTCs from tumor focus to peripheral blood is very rare in patients with early cancer, due to which detection of them will be very difficult. While the concentration of cfDNA in plasma is around 50 times more in patients with early cancer than in healthy people, which is easy to detect. Due to specific molecular and cellular characteristics of CTCs, they are difficult to capture and analyze.

1. 3. Circulating tumor DNA (ctDNA)

Detection of ctDNA is quantified using various technologies to determine either the mutant allele concentration (copies/ml) or the mutant allele fraction. Mostly, blood plasma is used for the analysis of ctDNA. The concentration of ctDNA in a sample is determined by digital polymerase chain reaction assay (dPCR). Digital PCR amplifies the concentration of small mutant molecules that are difficult to detect normally, through hundreds or even millions, to make quantification easy. The concentration of ctDNA in the plasma is helpful for staging and prognosis, as it has been shown to correlate with tumor size and stage of disease. The higher concentrations of ctDNA are correlated with poorer clinical outcomes at diagnosis and monitoring longitudinally throughout treatment of cancer. The lower concentration of ctDNA is present in the early stages of cancer, which makes the diagnosis difficult. The utilization of ctDNA for earlier detection of cancer is important for prevention of metastatic spread and increasing survival rates in cancer patients.

FDA approved liquid biopsy tests for early detection and monitoring of cancer

The short half-life of cfDNA allows immediate correlation with tumor cell status and offers continuous monitoring of cancer burden during treatments. Detection of MGMT hypermethylation (O⁶-methylguanine-DNA-methyltransferase) in cfDNA can predict the improvement in response and time to progression after treatment in patients with glioblastoma multiforme or metastatic colorectal cancer.

The liquid biopsies are being developed for a variety of purposes, like early detection, insights on prognosis and treatment selection. There are some FDA approved liquid biopsy tests available –

1. CELLSEARCH CTC kit in 2013 – To monitor patients with advanced metastatic breast, colon, and prostate cancers based on the CTCs concentration in plasma and other body fluids.

2. Cobas EGFR Mutation Test in 2016 – It was the first cfDNA based genetic test approved by the FDA as a companion diagnostic to detect epidermal growth factor receptor (EGFR) gene mutations in patients with non–small cell lung cancer (NSCLC).

3. Guardant360 CDx and FoundationOne Liquid CDx in 2020 – The FDA approved two comprehensive genomic profiling liquid biopsy tests. They can detect genomic alterations in multiple solid tumors from cell-free ctDNA of the plasma of patients and, with the help of NGS technology, identification of tumor mutations and selection of personalized medicine is also possible.

Gaurdant360 CDx is a companion diagnostic test for osimertib based patients with EGFR-mutant NSCLC. Apart from EGFR-mutant, it also provides information on other gene mutations like KRAS and BRAF. Later, the FDA granted approval for expansion of indications of this test including patients with NSCLC with EGFR exon 20 insertion mutations in plasma who may on treatment with amivantamab treatment and patients with KRAS G12C mutations who may on treatment with sotorasib.

FoundationOne Liquid CDx is approved as a companion diagnostic test for the poly (ADP ribose) polymerase inhibitor rucaparib for the treatment of advanced metastatic prostate cancer with BRCA1/2 mutations and three tyrosine kinase inhibitors like gefitinib, osimertinib and erlotinib for the first-line treatment of EGFR-mutant NSCLC. Later, FoundationOne Liquid CDx got approval by FDA for additional indications which include identification of mutations in BRCA1/2 genes in ovarian cancer with rucaparib treatment, identification of ALK rearrangements in NSCLC and identification of mutations in the PIK3CA gene in breast cancer with alpelisib treatment.

Liquid biopsy tests on the way to get FDA approval

There are currently various liquid biopsies in development for providing accurate information in the field of oncology. The LUNAR-2 test and Galleri test are two liquid biopsy early-detection tests which are in the process of getting FDA approval. LUNAR-2 test is a reliable and accurate first line less invasive blood test for colorectal cancer diagnosis at stage 1 and it will remove the colonoscopy and stool based testing at the very first phase of diagnosis. The Galleri test has the ability to detect more than 50 types of cancer, including pancreatic cancer for which there is no screening test available.

Early detection and monitoring of cancer has been shown to dramatically improve patient survival rates and quality of life, as well as significantly reduce the cost and complexity of cancer treatment, especially in particular for tumors that do not have a screening test available.

Reference

1. Chen M and Zhao H. 2019. Next-generation sequencing in liquid biopsy: cancer screening and early detection. Hum Genomics. 13(1):34.

2. Jia S et al. 2021. Values of liquid biopsy in early detection of cancer: results from meta-analysis. Expert Rev Mol Diagn. 21(4): 417-427.

3. Huiyan Luo et al. 2021. Liquid Biopsy of Methylation Biomarkers in Cell-Free DNA. Trends in Molecular Medicine. 27(5). https://doi.org/10.1016/j.molmed.2020.12.011.

4. Bossmann SH. 2020. Liquid biopsies for early cancer detection. InBiomaterials for Cancer Therapeutics. 233-259. Woodhead Publishing.