Liquid Biopsy: The Future of Diagnosis

Why does cancer become so fatal? What can we do to reduce its severity? are the two questions this post will try to answer. In the previous posts, we have discussed what causes cancer and our bodies defence against it. Now its time to talk about ways to diagnose it.

The traditional method is an invasive process involving the extraction of a small sample of tissue from the tumour suspected area, which is identified through performing scans. Using a specialised syringe, a small sample is extracted and DNA from that sample is sequenced to look for potential driver mutations. It is important to note that not all of the DNA is sequenced but only genes that could potentially cause the suspected type of cancer.

These mutations can broadly be classified into two types:-

1)Germline- Any detectable mutations present in germ cells.

2)Somatic- Seen in cancer cells and is of varying frequency, thus we need to sequence the DNA several times.

This process comes with a lot of limitations, of which some are listed below.

1) Being an invasive process it may not be feasible to carry out based on the location( e.g-Lungs or Brain), and size of the tumour.

2)The DNA that can be extracted might not be enough for the increasing request of molecular tests.

3)The DNA extracted might not completely reflect tumour heterogeneity or tumour evolution. (Drug treatments produce molecular changes in tumour cells)

The biggest disadvantage of this process, which answers our first question, is by the time diagnosis is complete it is usually too late. The tumour becomes malignant and is capable of performing metastasis. It becomes hard to contain the rapid proliferation of tumour cells.

To elaborate, it is difficult to extract tissue in case the tumour is the Brain or some other deep organ like the pancreas where it is difficult to detect the presence of tumour through a scan. Even if we can reach the tissue where the tumour is present, we still might not be able to detect tumour mutations as cancer cells are heterogeneous. If everything goes right, still we will only be able to get an inadequate sample from the tissue.

To substitute this technique we came up with LIQUID BIOPSY- which uses circulating DNA present in the blood to detect mutations. This DNA is assumed to be a representative of the whole genome. We can detect ct DNA (circulating tumour DNA) and confirm the presence of tumour even before it is visible in scans, which can save the patient some critical time.

Circulating cell-free DNA (cfDNA) is DNA shed from normal or tumour cells, by apoptotic or necrotic processes, into the bloodstream. Circulating tumour DNA (ctDNA) is a subset of cfDNA representing up to 1% of the total cfDNA. Patients with cancer have been reported to have higher levels of cfDNA than healthy individuals (ranges from 0 ng/mL to 1000 ng/mL). ctDNA can be identified and differentiated from cfDNA

• By fragments size and/or variations in the genetic abnormalities compared with normal cells. (Generally, ctDNA fragments are shorter than the somatic DNA fragments in plasma)

• By the presence of genetic variations found in cancer. (These genetic variations can be identified using genomic technologies such as digital polymerase chain reaction (dPCR) or next-generation sequencing (NGS).

Clinical Need for Liquid Biopsy

A.Cancer Diagnosis

1. Earlier diagnosis of cancer would enable treatment to be initiated sooner, and curative surgery may be carried out if the tumour is diagnosed at an early stage.

2. Sensitive and specific cancer detection may speed up the time to diagnosis and treatment for symptomatic patients.

3. Screening for markers of disease at the population level could enable early intervention in presymptomatic individuals.

B.Prognosis, residual disease and risk of relapse

After treatment with curative intent, the identification of patients with residual disease who are at high risk of relapse may be used to stratify patients to adjuvant therapy. Effective stratification spares low-risk patients from overtreatment.

C.Treatment selection

1. Introduction of novel molecularly targeted and immunotherapy agents necessitate improved tools for molecular profiling of patients and for treatment stratification.

2. Tumor biopsies could lead to false-negative results and suboptimal therapy selection.

D.Monitoring disease burden

1. Currently treatment monitoring is performed using imaging or molecular methods have limited accuracy, or are associated with a logistical burden or radiation exposure. The ideal monitoring assay should be repeatable serially over time, with minimal risk to patients, and should provide an accurate read-out of tumour burden.

Timeline of Development