Evolving role of liquid biopsies in management of breast cancer

Written by Mary N. O’Reilly & Michaela J. Higgins, St. Vincent’s University Hospital

Summary:

Liquid biopsies have the potential to become a gold standard method of diagnosing and monitoring those with breast cancer in the near future. The wealth of information that liquid biopsies can provide has numerous applications including diagnostic, prognostic, therapeutic response prediction and detection of relapse in those who were treated with curative intent. This review will discuss the role of three potential biomarkers for breast cancer which can be obtained from liquid biopsies; circulating tumour cells, circulating tumour DNA and exosomes.

 

Introduction

Breast cancer is the most common cancer among females (after skin cancer) in the world. In recent years, large scale clinical trials of new interventions and treatments have led to significantly improved diagnosis, therapy and survival for breast cancer patients.  We are moving away from traditional cytotoxic chemotherapy in favour of more targeted and therefore potentially less toxic therapies. Precision oncology describes efforts made to determine and target the drivers of a cancer at a molecular level. To date, most analyses of tumours have been performed on histopathological tissue samples. In contrast, so called ‘liquid biopsies’ have the potential to facilitate diagnosis, prognosis, measurement of tumour burden and response to therapy via simple, non-invasive blood sampling. (Figure 1) Liquid biopsy has the potential to inform the treating physicians decisions in a number of ways including decision to omit or include adjuvant therapy after surgery by detecting minimal residual disease, detecting early recurrence and analysis of response to disease so the treatment can be altered if it is not effective. Here we review the most common types of ‘liquid biopsies’ available; namely, circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and exosomes. We will discuss how they are currently used and their potential future applications.

 

Figure 1: Information which can be obtained from a liquid biopsy. Created using www.biorender.com

 

Methods of biopsy

Tissue biopsy is the traditional method of diagnosing cancer. While this is the best practice and continues to be standard of care for patients, it has considerable drawbacks. Tumours may present in challenging locations such as lung, liver or bone which cannot easily be accessed without interventional radiology expertise and space[1]. For the patients, there are associated risks of pain, infection, bleeding and damage to local structures depending on the area which needs to be biopsied. Liquid biopsy by contrast, takes the form of a small volume (approx. 10 ml) venous blood sample which can be collected in a low cost, minimally invasive manner[2]. After diagnosis and treatment commencement, it can be used in conjunction with tissue biopsy to assess response to treatment and disease relapse. Table 1 compares the advantages and disadvantages of these approaches.

 

 

Tissue Biopsy		Liquid Biopsy
Advantages	Disadvantages	Advantages	Disadvantages
Confers essential histological confirmation which remains the gold standard for cancer diagnosis	Cancers are heterogenous and a tissue biopsy only reflects one portion of the tumour	Potential to reflect the overall genomic landscape of the tumour	Liquid biopsy technologies available predominantly in the research setting and require laboratory expertise which is not yet routinely available. The technologies themselves are evolving rapidly
	Can be difficult to access depending on site of tumour	Easily collected peripheral blood sample	Unable to detect histological tissue type from which the tumour arises
	Resource-intensive – expertise to perform the procedure, operating room and nursing staff	Less risk of patient morbidity related to an adverse event from the biopsy	May not be sensitive enough to detect low volume disease eg detection of minimal residual disease or early recurrence
	Invasive/painful for the patient	Can be serially collected to reflect tumour molecular changes over time eg to identify emergence of resistance to treatment	May be insufficient tissue for next generation sequencing
	Long turnaround time – a number of weeks for next generation sequencing	Rapid turnaround time- 7-10 days for next generation sequencing results
	Challenges with preparation and storage of samples may impact biomarker interpretation eg ER status of decalcified bone sample	Can be used as a complementary diagnostic tool for molecular analysis

Table 1: Comparison of liquid and tissue biopsies

 

Circulating tumour cells

CTCs are cells which have entered systemic circulation after dislodgement from the primary tumour[3]. Some primary tumours shed millions of cells a day but only a tiny proportion of these will develop as a distant metastasis[4]. Identification of the cell surface molecule epithelial cell adhesion molecule EpCAM[3] is the most commonly used technique to identify CTCs. Tumour biomarker profiles, such as the HER2 receptor and oestrogen receptor status which are used to guide treatment, can change over the disease course thus sampling of a new metastasis is recommended[5]. Obtaining a repeat biopsy is not always possible for a number of reasons such as deteriorating patient condition or a difficult to access tumour location. CTC examination has the potential to not only guide treatment decisions which will result in a better prognosis, but also to spare breast cancer patients the potential toxicities of treatments to which their cancer has become resistant. The DETECT III trial analysed patient who had HER2-positive CTCs but HER2-negative metastatic breast cancer (by prior tissue sample). Patients were randomised to receive either standard treatment or standard treatment with lapatinib[6] which is a small molecule tyrosine kinase inhibitor targeting HER2. The study showed a survival advantage among patients who received lapatinib thus suggesting that CTC-driven change in therapy was useful and that the detection of HER2positive CTCs was predictive of benefit for patients. CTCs can also be useful to guide prognostication in breast cancer. A pooled analysis of the phase II multicentre trials BEVERLY -1 and 2 showed that patients who had detectable CTCs at baseline had a shorter overall survival compared with those who did not [7]. The SUCCESS A trial showed that in high risk early breast cancer the presence of CTCs two years after completion of chemotherapy was associated with decreased overall survival [8]. The IMENEO study was a large international meta-analysis which analysed the clinical validity of CTC count in early, non-metastatic breast cancer patients who received neoadjuvant therapy.  Data from 2,156 patients was collected and again it was found that those who had CTC detected at baseline had worse overall survival outcomes[9].  Gao et al [10] analysed whether CTC could be used in breast cancer screening. They found that use of combined imaging, ultrasound or mammogram with CTC increased the accuracy of breast cancer diagnosis.

 

Although there has been great enthusiasm among patients and physicians to adopt routine usage of CTCs, there are a number of limitations. The studies above suggest that at present CTCs should be utilised as a complementary test performed to add information to other examinations. A significant limitation is the fact that CTC detection is based on identification of epithelial markers, which are sometimes lost. Some CTCs undergo transformation from epithelial to mesenchymal cells. This transition increases the cells ability to establish peripheral micrometastases[3]. These cells become undetectable by several commonly used detection methods[11]. The laboratory techniques used to detect CTCs and their sensitivities are very much in evolution and improving[12].

 

Circulating tumour DNA

ctDNA is tumour- specific free DNA fragments released during apoptosis from the primary tumour or associated metastatic deposits which can serve as a biomarker for diagnosis, prognosis and prediction[13]. It is thought that ctDNA is released by all tumour sites; including metastases and therefore it may provide invaluable information with regards to tumour heterogeneity. Tumour-specific mutations allow the tumour DNA to be differentiated from other non-specific free DNA which originates from normal cells. There are two ways in which ctDNA is currently used for therapeutics[14]; the first is using a targeted approach to detect specific mutations in genes which are actionable such as PIK3CA and ERBB2. The other method is an untargeted approach where whole genome sequencing is performed on the DNA. This yields a vast quantity of information only a small fraction of which may be actionable.

CtDNA can detect occult tumours and therefore has the potential to become gold standard for diagnosis to detect the presence of tumour DNA before it is identifiable on imaging. The first custom built next generation sequencing based ctDNA test, Signatera was developed in 2019. This technology set out to detect early disease recurrence after surgery as well as detection of post-operative minimal residual disease. Coombes et al demonstrated that this test could identify disease relapse up to two years earlier than imaging in patients with early- stage breast cancer[15]. A number of studies have described concordance between primary tumour and ctDNA. Adalsteinsson et al analysed samples from 41 patients with either breast or prostate cancer, they found 88% concordance of clonal somatic mutations between ctDNA and matched tumour biopsies[16].

 

The predictive value of ctDNA was evaluated in a pooled analysis of MONALEESA2,3 and 7 trials[17]. The ctDNA from 1507 oestrogen receptor positive, HER2-negative metastatic breast cancer patients were analysed by next generation sequencing. Tumours harbouring mutations in FRS2, PRKCA, MDM2, ERBB2, AKT1 and/or BRCA1/2 identified by ctDNA were associated with improved progression free survival with the CDK4/6 inhibitor ribociclib in combination with endocrine therapy when compared to endocrine therapy alone. Dawson et al [18]performed a proof of analysis study where they assessed the presence of ctDNA in patients with metastatic breast cancer and somatic genomic alterations. ctDNA was detected in 97% (29/30) of patients. 20 patients had serial assessment of ctDNA in their plasma to asses response to treatment.  19 of these patients (95%) had fluctuations in the levels of ctDNA detected which reflected response to therapy seen on imaging.

 

Circulating Tumour DNA		Circulating tumour cells
Advantages	Disadvantages	Advantages	Disadvantages
Allows for detection of presence of cancer even in cases of low tumour burden	Proteomic and metabolomic analysis not possible	Proteomic and metabolomic analysis can be used to provide additional information	Heterogenicity of CTCs can make interpretation complicated
Can be used to monitor response to therapy as well as development of resistance to therapy	Low signal to noise ratio in early stage disease	Allow structural evaluation of cancer phenotype	Not routinely available at present outside of research setting in Ireland. Work is needed to improve sensitivity and specificity of this test before they are incorporated into clinical practice
	Not routinely available at present outside of research setting in Ireland. Work is needed to improve sensitivity and specificity of this test before they are incorporated into clinical practice	Amenable to immunolabelling techniques

Table 2: Comparison of ctDNA and CTCs

 

 

Exosomes

A further type of liquid biopsy in development for breast cancer is based on exosomes. Exosomes are extracellular vesicles with a diameter ranging from 50 to 150nm. They are released from their cell of origin, enveloped in a lipid bilayer containing molecular constituents from their cell of origin such as DNA, RNA, proteins and metabolites [19]. Exosomes are stable and plentiful in bodily fluids. Patients with breast cancer have been shown to have substantially higher levels of exosomes in their blood than matched cancer free controls[20]. Given this distinction, there may be a role for the use of exosomes in the detection of breast cancer. Exosomes from malignant cells have a different microRNA (miRNA) and protein profile compared to normal cells allowing analysis of the tumour status and potentially treatment response in vitro at a particular or serial timepoints[21]. Exosomes are taken up by receiver cells which can translate the miRNA into protein identical to the original cancer cell. This method of communication confers phenotypic traits of the cancer to previously heathy cells. In this way some exosomes play a role in mediating cancer progression and metastasis.

 

Conclusion

The potential for the widespread clinical use of liquid biopsies in the detection and management of modern breast cancers is huge and attractive to patients and their treating physicians. Liquid biopsies have various applications across many aspects of the breast cancer journey from molecular profiling at diagnosis which will guide therapeutic decisions; including identification of those who should receive adjuvant therapy to surveillance and early detection of occult metastases. Clinical studies suggest that several techniques measuring in particular circulating tumour cells or cell free tumour DNA, are sensitive, reliable and may rapidly inform treatment of patients with breast cancer. We anticipate and hope that these assays will become available to Irish patients outside the research setting in the next few years.

 

 

References: 

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