This article delves into the evolution and applications of Liquid Biopsy (LB) in Oncology, showcasing its historical context, procedural principles, and its pivotal role in detecting and characterising cancer through the analysis of circulating biomarkers in body fluids. 

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The conventional usage of the term biopsy in the oncology field refers to the microscopic examination of tissue samples for confirmation and characterisation of cancer.

Liquid Biopsy (LB) tests various biological fluids like blood, urine, saliva, CSF, pleural fluid etc for the detection of cancers.

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Background of Liquid Biopsy (LB)

Mandel and Metais in 1948 showed the presence of circulating free DNA (cfDNA) in human blood. Dennis Lo showed the presence of fetal Y chromosomes in the blood of pregnant mothers carrying male babies that helped in identifying point mutations, aneuploidy and genetic anomalies.

In 1977 it was reported that levels of cfDNA were higher in the blood of cancer patients than in healthy individuals. These cf DNA had peculiar genetic mutations that help in the characterisation of cancer and the detection of specific molecular anomalies and are termed as ct DNA.

The first FDA approval for LB was given in 2016 known as the ctDNA-based companion diagnostic test for lung cancer.

LB is easy to carry out and is reproducible over time. LB discerns specific cancer-related mutations that in turn help in therapeutic choices and also help in monitoring treatment response. The main and only drawback of LB is low yield due to the limited amount of biomarkers in body fluids.

The circulating biomarkers in body fluids are circulating cell-free tumour DNA (ctDNA), circulating tumour cells (CTCs) or circulating RNA c RNA, proteins, and lipids constituting exosomes, and Tumour Educated Platelets (TEP).

By detection of circulating biomarkers, LB helps in the detection of cancers as well as genetic changes associated with particular cancer that in turn has both prognostic predictive and therapeutic importance. It has been estimated that one millilitre of blood contains 25 ng of cell-free DNA. Further, a tumour of 100 g sheds about 3.3% of ct DNA daily.

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Principe of Liquid Biopsy (LB):

Blood withdrawal (EDTA tubes by qualified personnel ) à transportation time up to 3 hours à separation of plasma (2 steps method): 

Low-speed centrifugation (1200- 1600 g) to avoid leukocyte lysis and second, high-speed centrifugation of the supernatant (3000 g) to remove all contaminants) à storage @ 20 C x 1 month or -80 C x à thawing à cf DNA (directly related to tumour burden) à ctDNA release.

• RT –PCR method
• Digital PCR
• NGS method à results

LB detection depends on:

• Amount of fluid taken
• Standard practices for proper collection and transportation
• Methodology for processing and storage of cfDNA
• Experience in laboratory services
• Burden of malignancy - The advanced stage of malignancy has a high concentration of ctDNA. Early-stage oligometastatic, brain or bone-only metastatic sites have low yields
• Type of sample taken. In CNS malignancy, CSF fluid has a high yield as compared to plasma

Usage of LB in different solid cancers:

1. Lung cancer – For newly diagnosed advanced NSCLC patients, LB helps in the detection of driver mutations like EGFR, ALK, ROS, RET, MET, BRAF, ERBB2, KRAS, MAP 2 K1, PIK3CA, TP 53. LB also detects acquired mutation exon 20 T 790 M EGFR mutations.

2. Breast cancer - LB helps genes frequently mutated in breast cancer like estrogen receptor 1 (ESR1), ERRB2, ERBB3, K RAS, TP 53, and importantly PI3K 3 CA. 

These markers have a direct correlation with the tumour burden. ESR1 gene predicts resistance to treatment with hormonal therapy. PIK3CA mutation occurs in 50 % of patients who progress on CD K 4/6 inhibitors where Alpelisib is used.

3. Colorectal cancer - LB detects ctDNA in colorectal cancer (CRC) that has prognostic value. LB also detects RAS and BRAF mutations that help in the decision for usage of targeted therapy.

LB also can be done for acquired resistance to anti-EGFR therapies for a rechallenge strategy in the evaluation of RAS mutational status on ctDNA in patients with RAS wild-type mCRC.

4. Melanoma - LB helps in the detection of BRAF and NRAS mutations in patients with melanoma and monitoring of ctDNA during treatment.

5. Monitoring of therapeutic response: LB helps in the detection of MRD in solid cancers by quantification of ct DNA levels.

6. LB in urine– In colon cancer was even used for the analysis of other solid tumours, including KRAS detection in patients with stage IV pancreatic cancer, and in stage III-IV CRC patients.

7. LB in saliva - Detect c-erbB-2 in saliva specimens of BC patients.

8. LB in CSF– Detects tumours of the central nervous system (primary or metastatic) due to the higher presence of cfDNA in CSF concerning matched plasma samples.

9. LB from pleural effusions – Detects EGFR sensitizing mutations by analysing the cfDNA extracted from pleural effusion.

10. LB in patients on immunotherapy - Detection of cfDNA/ctDNA, CTCs for patients on immunotherapy show less response to treatment.

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Clinical scenarios

A. 82-year-old male, Mr X, with co-morbidities of COPD, coronary artery disease, hypertension, and type II diabetes had a clinical history of cough, breathlessness oxygen dependent weight loss and decreased appetite for 2 months. He was evaluated by a pulmonologist and cardiologist.

Examinations

1. Chest X-ray showed multiple small opacities in both lungs.
2. 2 D Echo showed EF 55% with no regional wall motion abnormalities. He was referred to a medical oncologist for further care.
3. PET CT W/B showed FDG avid multiple lesions in both lungs, FDG avid lesion in the left adrenal gland and D 11, D 12 and multiple ribs. CE MRI brain showed age-related atrophy.

Management

1. The patient was symptomatic for his disease and refused for tissue biopsy.
2. Family members were explained the clinical scenario and the option of LB was given.
3. A Liquid Biopsy was ordered that showed the presence of malignancy that was EGFR mutation-positive, He was started on Tab. Osimeritinib 80 mg once a day.
4. After 2 weeks he showed significant clinical response and was off oxygen.
5. After completion of 3 months of Tab Osimeritinib, PET CT was repeated and showed near-completed resolution of lung and skeletal lesions.
6. Currently, the patient is on Tab Osimeritinib 80 mg for 9 months.

B. A 42-year-old female, Mrs Y was diagnosed with stage IV carcinoma breast diagnosed three years back. Her medical records revealed her breast biopsy showed IDC with IHC showed ER = 8/8/, PR =8/8, Her 2 = 0 and was on Tab Ribociclib 600 mg + Tab Anastrazole 1 mg for the past years. She now started with complaints of increasing fatigue and bony pains.

Examinations

PET CT was done that showed features of new FDG avid skeletal lesions and subcentimeter FDG avid lung lesions.

Unfortunately, her biopsy sample was not preserved. A Liquid Biopsy for PIK 3 CA mutation was done that came positive.

Management

1. She was started on Tab Alpelisib 150 mg BD and Inj Fulvestrant 250 mg + 250 mg I/M. After 1 month, she showed clinical improvement. PET CT showed a metabolic response to lung lesions.
2. The patient is currently on Tab Alpelisib 150 mg BD and Inj Fulvestrant 250 mg + 250 mg I/M for 6 months with normal ADLs.

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Disclaimer- The views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of M3 India.

About the author of this article: Dr Imran Khan is Dr NB Medical Oncology, ECMO Associate Consultant Oncology, Fortis Escorts Hospital, Amritsar.