Key words
liquid biopsy - breast cancer - circulating tumour DNA - circulating tumour cell -
targeted therapy
Introduction
In the last century, scientific and clinical advances in the diagnosis and treatment
of breast cancer have resulted in a significant paradigm shift. The so-called “Halsted
Doctrine”, which viewed breast cancer as a local event with healing only possible
by the most radical surgery, was replaced by the “Fischer Doctrine”, which regards
even early stage breast cancer as a systemic disease [1]. Today we know that haematogenous dissemination already takes place in very early
stages of breast cancer and that the circulating tumour cells (CTCs) will even be
found in patients with pre-invasive breast lesions [2]. At the same time the hypothesis of metastatic inefficiency states that most of
these cells are eliminated by the immune system or the mechanical shear forces in
the blood [3], [4]. Only a small CTC subpopulation can persist for long time in the blood
or other “homing sites”, such as the bone marrow, and is considered a surrogate
marker of minimal residual disease (MRD). These cells also exhibit long-term tumour
cell dormancy and may be detected in the peripheral blood even many years after the
initial diagnosis of the disease [5].
It still remains unclear which characteristics contribute to certain CTCs being able
to “wake up” and survive several steps of the metastatic cascade in order to later
evolve into distant metastases. One theory currently under discussion states that
these cells are particularly aggressive and undergo the so-called epithelial-mesenchymal
transition (EMT). This process involves a number of changes, for example, the loss
of polarity and intercellular adhesion, an increase in mobility and invasiveness,
and finally the loss of the epithelial, and acquisition of the mesenchymal, phenotype
[6]. Furthermore, EMT can generate cells with stem cell characteristics that have resistance
mechanisms as well as a very high potential of self-renewal and may be regarded as
actual precursors of distant metastasis [7], [8].
The Concept of Liquid Biopsy
The Concept of Liquid Biopsy
In breast cancer patients peripheral blood samples may contain not only circulating
tumour DNA (ctDNA) and/or RNA fragments (non-coding RNA, ncRNA) but also intact CTCs.
The ctDNA/RNA fragments are released continuously into the bloodstream by the primary
cancer and/or the metastatic lesion itself and by the decaying CTCs. Liquid biopsy
is defined as the detection and analysis of these blood-borne biomarkers in cancer
patients. Thus, in heterogeneous tumours such as breast cancer, oncogene mutations
and amplifications may be detected more effectively than with tissue biopsies, which
only represent a limited tumour area [9], [10]. Further benefits compared to tissue biopsy include the possibility of serial analyses
by simple blood sampling and the detectability of multiple or hard to reach metastases.
In addition, due to its low invasiveness liquid biopsy acceptance is very high among
patients. This paper
presents the current data on clinical significance as well as the potential applications
of liquid biopsy in primary and metastatic breast cancer.
Detection Techniques
Circulating tumour cells
In principle CTC detection involves two independent steps: Isolation of the cells
from the whole blood and their actual detection. Isolation of these cells from the
other constituents of the blood draws on various cell characteristics, mainly physical
(size- and/or density-based) and biological (specific antigen expression). Examples
include the so-called density gradient technique with Ficoll and antigen-based modalities
with epithelial (e.g., EpCAM, cytokeratins) and/or tumour-specific markers such as
CEA or HER2. In antibody-based isolation, the specific antibodies are mostly coupled
to magnetic particles, with the enriched cells then being separated by a magnetic
field for further analysis.
The subsequent CTC detection may then be based again on nucleic acid (RT-PCR, qRT-PCR,
multiplex RT-PCR) or the antigen characteristics (immunocytochemistry, immunofluorescence,
immunofluorescence flow cytometry) of the CTCs. While the sensitivity and specificity
of nucleic-acid based detection is rather high, their drawback is CTC lysis during
the analysis, making it impossible to assess cell morphology or perform further cell
analysis.
The most common technique for CTC detection in clinical trials is the CellSearch system
(Menarini Silicon Biosystems, Inc.), which has been approved by the U. S. Food and
Drug Administration (FDA) for metastatic breast, colon and prostate cancer. With this
standardised assay, EpCAM-positive CTCs are isolated immunomagnetically from 7.5 ml
whole venous blood and subsequently stained by immunofluorescence with antibodies
against cytokeratins (CK) 8, 18, 19, the specific leukocyte marker CD45, and the nuclear
marker DAPI. The sample is then scanned by a semi-automatic fluorescence microscope
and the potential CTCs are automatically imaged. Finally, the analysis is performed
by an experienced examiner: CK 8/18/19 and DAPI-positive as well as CD45-negative
cells with specific morphology are identified as CTCs ([Table 1], [Fig. 1]). The CTCs detected by the CellSearch system can be harvested by
micromanipulation and are available for further analysis (e.g., single-cell gene
expression analysis). The downside of this technique is on the one hand the subjective
assessment by the examiner, and on the other hand also the fact that it is an EpCAM-based
technique and the cells cannot be detected after EMT due to the loss of epithelial
characteristics [11], [12].
Table 1 Examples of typical cytomorphological criteria for the identification of isolated
tumour cells (based on [13]).
|
Cell morphology/phenotype
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Fig. 1 Detection of isolated tumour cells by various techniques: a Apoptotic tumour cell stained by immunocytochemistry (M30 antibody [AK]); b Vital tumour cell stained by immunocytochemistry (anti-cytokeratin [CK]-AK); c Immunofluorescence, double staining (anti-CK-AK: green, anti-oestrogen receptor-AK:
red); d Immunofluorescence-stained tumour cell cluster (anti-CK-AK).
Another challenge in CTC diagnostic work-up is the low detection rate, primarily in
non-metastatic patient populations. Particularly in view of the known heterogeneity
of the tumour, it is advisable to analyse as many CTCs of a patient as possible. In
this context, many enrichment/isolation techniques have been explored in recent years
to increase the detection rate. Another possible approach is the analysis of larger
blood volumes, for example, as part of diagnostic leukapheresis (DLA). On average,
this extracorporeal filtration procedure will filter 2770 ml of blood. The CTCs are
isolated by density gradient and an aliquot of the DLA product is then analysed with
the CellSearch system. This significantly increases the CTC detection rate and number
of CTCs identified [14]. However, analysis of large blood volumes requires considerable time on the part
of the patient: the procedure takes about one hour. Lab panel changes after DLA have
also been observed, e.g., slight decreases in leukocyte and haemoglobin counts,
though these are not clinically significant. It remains to be seen to what extent
the collection of several thousand CTCs will have an impact on clinical significance
and whether DLA will improve CTC-based diagnostic work-up.
Circulating tumour DNA
The term circulating tumour DNA (ctDNA) refers to free DNA fragments, originating from the tumour (primary tumour, metastasis,
isolated tumour cells), which can be detected in blood and other body fluids. This
must be distinguished from the broader term cell-free DNA (cfDNA) or free circulating DNA (fcDNA), which refers to all non-tumour specific cell-free DNA fragments. Decaying normal
cells can also release cfDNA into the blood. While the older trials primarily focused
on the detection of the total amount of freely circulating DNA in the blood, more
recent studies have addressed the specific detection techniques for ctDNA.
Detection is usually carried out on blood plasma. Detection is determined by the percentage
of ctDNA compared to cell-free DNA from normal cells. Since this percentage may be
quite small, ctDNA detection requires highly sensitive techniques. These include digital-droplet
PCR (ddPCR), beads amplification magnetics-PCR (BEAMing-PCR), and digital next-generation
sequencing (dNGS) [15], [16], [17]. Here, ddPCR and BEAMing-PCR are the so-called targeted detection techniques (targeted
approach), in which only a few gene loci can be examined simultaneously. In other
words, the search is targeted at specific, already known tumour mutations, e.g., in
genes PIK3CA, ESR1, AKT1, ERBB2 and PTEN. The term dNGS, on the other hand, summarises
several non-targeted gene analysis techniques (untargeted approach), where large DNA
molecules are sequenced and thus numerous unknown genetic
alterations and mutations can be detected. These include, for example, array
CGH (array-comparative genomic hybridization), whole-genome sequencing and exome sequencing.
These techniques usually identify gene mutations that may contribute to the assessment
of developing resistance [15], [16].
Clinical Applications of Liquid Biopsy in Early Breast Cancer
Clinical Applications of Liquid Biopsy in Early Breast Cancer
Improved prediction of prognosis
Individual tumour cells reaching the blood vessels and their subsequent dissemination
are important steps in the metastatic cascade. According to studies, pre-invasive
lesions of the breast, such as ductal carcinoma in situ (DCIS), can be accompanied
by tumour cell dissemination [2], [18], so that haematogenous tumour cell dissemination is considered an early event in
the course of malignant disease. The CellSearch system can detect circulating tumour cells in 20 – 30% of patients with non-metastatic breast cancer [19] ([Table 2]). In a pooled analysis, Janni et al. were able to evaluate the data of 3173 patients
with stage I – III breast cancer and demonstrated that the detection of at least one
CTC in 7.5 ml blood predicts a significantly worse clinical outcome [19]. CTC-positive patients were twice as
likely to die from breast cancer as women who did not have CTCs at the time of
diagnosis (hazard ratio [HR]: 2.04; 95% CI: 1.52 – 2.75). CTC-positivity also correlated
with a statistically significant shorter overall survival (HR 1.97; 95% CI: 1.51 – 2.59),
disease-free survival (HR 1.82; 95% CI: 1.47 – 2.26) and distant disease-free survival
(HR 1.89; 95% CI: 1.49 – 2.40).
Table 2 Prognostic significance of circulating tumour cells in breast cancer: the most important
trials.
|
Trial
|
Number of patients
|
Stage
|
Positivity rate
n (%)
|
Assay
|
Correlation with prognosis
|
|
1 defined as ≥ 1 CTC per 7.5 ml
2 defined as ≥ 5 CTCs per 7.5 ml
Acronyms: BCSS – breast cancer-specific survival; DDFS – distant disease-free survival;
DFS – disease-free survival; LRRFS – loco-regional recurrence-free survival; OS –
overall survival; pCR – pathological complete response
|
|
Janni et al. [19]
|
3173
|
Stage I – III
|
641 (20%)1
|
CellSearch
|
DFS, DDFS, BCSS, OS
|
|
Cristofanilli et al. [20]
|
2436
|
Stage IV
|
1099 (45.1%)2
|
CellSearch
|
OS
|
|
Bidard et al. [21]
|
1574
|
Stage I – III before neoadjuvant chemotherapy
|
398 (25.2%)1
|
CellSearch
|
OS, DDFS, LRRFS; pCR rate higher in case of CTC positivity (24.2% vs. 17.4%)
|
While large meta-analyses ([Table 2]) have confirmed the prognostic relevance of CTCs, the existing data on circulating DNA is much less extensive ([Table 3]). A meta-analysis published in 2018 by Tan et al. included a total of 1127 patients
from 10 trials, the majority with non-metastatic disease [22]. Unlike in the pooled analyses on CTC significance, the populations here were markedly
smaller (336 patients maximum). In addition, the heterogeneity of the different assays
used hampered direct comparison of the trials considerably. Some of the trials analysed
the total amount of cfDNA, while the others studied the detection of pre-defined genomic
alterations. Despite these shortcomings, the meta-analysis suggests a possible prognostic
significance of cfDNA and mutation detection in non-metastatic breast cancer.
Table 3 Prognostic significance of circulating DNA in breast cancer patients (based on: [22], only trials with at least 100 patients were included).
|
Trial
|
Number of patients
|
Setting
|
Technique:
|
Prognostic significance: OS
|
|
1 Serum
2 Plasma
Acronyms: dPCR – digital PCR; HR – hazard ratio; OSMSP – one-step methylation-specific
PCR; PCR-SSCP – PCR-single-strand conformation polymorphism; RFS – relapse-free survival
|
|
Fujita et al. [23]
|
336
|
Stage I – II
|
OSMSP1
Met-DNA (±)/Total cfDNA (high/low)
|
OS:
Yes (HR for Met-DNA 3.17; for total cfDNA 4.03)
DFS/RFS:
Met-DNA: No (HR 2.3)
Total cfDNA: Yes (2.70)
|
|
Fernandaz-Garcia et al. [24]
|
194
|
Stage IV
|
TaqMan, RT-PCR
Total cfDNA (high/low)
|
OS:
yes (HR 2.296)
|
|
BRE12–158 [25]
|
151
|
Stage I – III
triple-negative, non-pCR
|
FoundationOne Liquid2
ctDNA (±)
|
OS:
yes (HR 2.7)
DDFS:
yes (HR 3.1)
|
|
Garcia et al. [26]
|
142
|
Stage I – III
|
PCR-SSCP2
ctDNA (±)
|
OS:
no (HR 1.60)
DFS/RFS:
yes (HR 2.70)
|
|
Fujita et al. [27]
|
120
|
Stage II – III
post-therapy
|
OSMSP1
Met-DNA (±)/Total cfDNA (high/low)
|
OS:
yes (HR for Met-DNA 4.91; for total cfDNA 4.11)
DFS/RFS:
yes (HR for Met-DNA 4.23; for total cfDNA 1.93)
|
|
Shaw et al. [28]
|
112
|
Stage IV
|
ddPCR2
Total cfDNA
|
OS:
yes (HR 2.20)
|
|
Shaw et al. [29]
|
110
|
Stage I – III
|
dPCR1
mut. PIK3CA (±)
|
OS:
no (HR 3.92)
DFS/RFS:
yes (HR 4.78)
|
Liquid biopsy treatment monitoring
Non-invasive diagnostic blood tests allow serial studies which can be repeated as
often as needed during treatment or after its completion. This provides unique insight
into the current cancer situation. Since systemic treatment exerts a selection pressure
on MRD, blood samples can help assess the persistent tumour cell population [30]. This way, it is possible to monitor the response to treatment and identify those
patients with increased risk of relapse who might benefit from additional therapeutic
approaches.
We now know that CTCs can persist beyond (neo)adjuvant therapy (so-called persistent CTCs, [Table 4]). The SUCCESS trial, initiated in Germany, was the largest analysis of CTC persistence
in non-metastatic disease to date [31]. In 2026 patients, CTCs were examined with the CellSearch system before the patients
started adjuvant chemotherapy. At least one CTC was detected in 21.5% of the women.
After completion of chemotherapy, a blood sample was taken from 1493 patients. The
positivity rate was 22.1%. The detection of CTCs prior to chemotherapy correlated
with clinical outcome (DFS, DDFS, BCSS and OS), but only to a limited extent with
CTC persistence. For example, 76 patients had a positive CTC status both before and
after chemotherapy. In 936 women both blood samples were CTC-negative. In 491 patients
the CTC status changed (+ → − in 238 cases and − → + in 253 cases). The detection
of persistent
CTCs predicted unfavourable clinical outcomes (DFS: hazard ratio 1.124, p = 0.02,
OS: hazard ratio 1.162, p = 0.06). This suggests the development of effective resistance
mechanisms by the tumour cells, allowing them to evade the effects of cytotoxic treatment
[21], [31]. In addition, neoadjuvant studies have shown that CTC response does not correlate
with primary tumour response to treatment.
Table 4 Clinical significance of persistent CTCs in early breast cancer.
|
Trial
|
Number of patients
|
Setting
|
Positivity rate
(%)
|
Correlation with survival
|
|
1 Analysed with CellSearch
2 Analysed with AdnaTest
Acronyms: DDFS – distant disease-free survival; DFS – disease-free survival; NACT
– neoadjuvant chemotherapy, OS – overall survival
|
|
Rack et al. [31]
|
1493
|
Stage I – III
N+ or high-risk N0
Blood test after adjuvant chemotherapy
|
22%1
|
yes: DFS, OS
|
|
Bidard et al. [21]
|
1200
|
Stage I – III
Blood test after NACT
|
15%1
|
yes: OS, DDFS
|
|
Riethdorf et al. [32]
|
207
|
high-risk
Blood test after NACT
|
11%1
|
not analysed
|
|
Kasimir-Bauer et al. [33]
|
133
|
Stage II – III
Blood test before and after NACT
|
8%2
|
no
|
Numerous smaller studies have explored the question of how the detection of circulating
DNA may complement treatment monitoring in early breast cancer ([Table 5]). Li et al. studied plasma samples before, during and after neoadjuvant chemotherapy
in 52 patients and demonstrated that ctDNA analysis after 2 cycles of treatment correlated
better with pCR probability than radiological diagnostic work-up [34]. Similar results were reported by Magbanua et al., who performed ctDNA ultra-deep
sequencing in 84 patients treated in the I-SPY 2 trial [35]. Within 3 weeks after treatment was started, the ctDNA positivity rate dropped sharply
from 73 to 35%. All patients who achieved pCR were ctDNA negative after chemotherapy.
In the non-pCR subset, the detection of persistent ctDNA after NACT predicted a significantly
increased risk of metastasis (hazard ratio 10.4).
Table 5 Clinical significance of circulating DNA during and after neoadjuvant therapy in
early breast cancer: Summary of the key trials.
|
Trial
|
Number of patients
|
Technique
|
Positivity rate (%)
|
Results
|
|
Acronyms: AUC – area under the curve; DFS – disease-free survival; ddPCR – digital
droplet PCR; dPCR – digital PCR; DDFS – distant disease-free survival; EFS – event-free
survival; NACT – neoadjuvant chemotherapy; NGS – next generation sequencing; OS-MSP
– one-step methylation-specific PCR; RCB – residual cancer burden
|
|
Takahashi et al. [36]
|
87
|
OS-MSP
|
23% before NACT
|
ctDNA persistence associated with RCB
|
|
Magbanua et al. [35]
|
84
|
Ultra-deep sequencing
|
73% before NACT
9% after NACT
|
Persistent ctDNA 3 weeks after start of NACT associated with pCR (pCR rate 17% vs.
48%, p = 0.012); ctDNA detection after NACT associated with DDFS
|
|
NeoALTTO trial [37]
|
69
|
Mutation analysis PIK3CA and TP53 (ddPCR)
|
41% before NACT
20% 2 weeks after start
5% after NACT
|
Persistent ctDNA 2 weeks after start of NACT associated with a lower probability of
pCR, but not EFS
|
|
Garcia-Murillas et al. [38]
|
55
|
dPCR, high-depth DNA sequencing
|
69% before NACT
19% 2 – 4 weeks postop.
|
ctDNA persistence 2 – 4 weeks postop. associated with DFS (hazard ratio 25.1)
|
|
Li et al. [34]
|
52
|
NGS panel of 1021 genes
|
48% before NACT (most mutations in genes TP53, PIK3CA, GAB2, and IRS2);
ctDNA persistence in 70% of initially ctDNA-positive pts.
|
ctDNA after 2 cycles predicted the pathological response (AUC 0.81); higher rate of
relapse in case of persistent ctDNA (50 vs. 33%)
|
|
Sharma et al. [39]
|
30
|
Methylation analysis (genes BRCA1, MGMT, GSTP1, stratifin, MDR1)
|
Methylation before NACT: 76% at least 1 gene; 53% (BRCA1), 37% (MGMT), 43% (GSTP1),
83% (stratifin), 60% (MDR1) (76%)
|
Tumour response associated with increased methylation before NACT and decreased methylation
after NACT
|
|
Moss et al. [40]
|
30
|
Methylation analysis
|
80% before NACT
|
marked decrease in cfDNA under NACT; cfDNA associated with pCR in last month of NACT
(p = 0.006)
|
Liquid biopsy potential in follow-up
After completion of the primary therapy, which usually comprises surgery and, depending
on the subtype and the extent of surgical treatment, radiotherapy and chemotherapy,
possibly in combination with targeted therapy, when trying to estimate the remaining
risk of relapse, the attending physician can nowadays only refer back to the characteristics
of the disease at the time of the initial diagnosis. There are no other tools available
allowing individualised risk estimation. On the other hand, during follow-up patients
have a strong desire to learn their chances of recovery. In this context some women
demand analysis of the classical tumour markers (e.g., CEA, CA 15-3), a step which
the current guidelines expressly discourage [41], [42]. Several trials have explored how liquid biopsy can improve the prediction of prognosis
in the follow-up of asymptomatic patients.
In 2018, the data from two large-scale adjuvant therapy trials were published, which
studied the significance of CTC testing 5 years post-diagnosis in translational subprojects ([Table 6]). The CellSearch system was used in both trials. Interestingly enough, the prognosis
in women with persistent CTCs was significantly poorer than in the CTC-negative population,
especially in the subgroup of HR-positive tumours. For example, the U. S. trial demonstrated
that the annual risk of recurrence was 21.4% when at least one CTC was detected, compared
to only 2% in CTC-negative women.
Table 6 Clinical significance of persistent CTC in follow-up.
|
Trial
|
Number of patients
|
Date of CTC analysis
|
Positivity rate (%)
|
Median follow-up period
|
Correlation with prognosis
|
|
ECOG-ACRIN E5103 [47], [48]
|
547
HER2-negative stage II – III
|
4.5 – 7.5 years post-diagnosis
|
4.8%
|
2.6 years
|
Risk of relapse 12.7 × higher in patients with persistent CTCs; risk of relapse per
patient/year in the HR-positive population: 21.4 vs. 2.0%
|
|
SUCCESS-A [49]
|
206
Stage I – III (high-risk)
|
median 62 months post-diagnosis
|
7.8%
|
1 year
|
In HR-positive population: risk of relapse higher in CTC-positive pts. (hazard ratio
5.95)
|
According to smaller trials the circulating DNA may also contribute to risk stratification in the follow-up of asymptomatic patients
[43], [44], [45], [46]. In a group of 101 women Garcia-Murillas et al. were able to establish that serial
ctDNA measurements can predict the risk of recurrence [44]. Blood tests were repeated every 3 months in the first year and then every 6 months
for 5 years. ctDNA detection was based on the somatic mutations in the tumour tissue,
which were analysed by dPCR in the blood. Patients in whom ctDNA was detected during
the course of chemotherapy experienced a markedly shorter recurrence-free survival
(hazard ratio 16.7, p < 0.001), with the first blood samples after completion of chemotherapy
being initially ctDNA-negative in most patients. Clinical recurrence or distant
metastasis was observed a median of 10.7 months after the first ctDNA-positive
blood test. Interestingly enough, since ctDNA progression did not correlate with the
presence of brain metastases, this location may remain “mute” in liquid biopsy-based
detection.
The EBLIS trial also confirmed the potential of ctDNA-based monitoring in follow-up
[45]. Here, in the first 4 years after completion of chemotherapy, blood tests were performed
every 6 months. ctDNA detection relied on the detection of individual tumour-specific
mutation signatures, which were established by analysing the primary tumours. Clinical
assessment of the course of the first 49 patients revealed that ctDNA detection occurred
a median 8.9 months before local or distant relapse.
It is still unclear how these findings can be implemented in routine clinical practice
and which diagnostic or therapeutic consequences might derive from a positive ctDNA
blood test result. In the British c-TRAK TN trial (NCT03145961), monitoring by ctDNA
testing is performed every 3 months after completion of therapy in triple negative
disease. Patients with persistent ctDNA are randomized to pembrolizumab or observation.
In Germany, the SURVIVE trial initiated by the Department of Obstetrics & Gynaecology
at Ulm University Medical Centre plans to study the significance of liquid biopsy
in follow-up.
Liquid biopsy-based therapeutic interventions
The adoption of blood-based biomarkers in the practice of early breast cancer has
been hampered to date by the uncertainty regarding the clinical consequences. One
of the few trials exploring possible CTC-based therapeutic intervention in non-metastatic
disease was the multicentre Treat CTC trial [50]. Here, patients with HER2-negative primary tumour and persistent CTCs after completion
of (neo)adjuvant chemotherapy were randomised to 6 cycles of trastuzumab or observation.
A total of 1317 patients were screened. CTCs were detected in 95 women, of which 63
were successfully randomised. Since the primary endpoint (successful CTC elimination
by trastuzumab) was not met, the trial was terminated. Nor did HER2-targeted therapy
improve clinical outcome: after a median follow-up of 13 months, invasive disease-free
and overall survival were the same in both arms. The precise inclusion criteria must
be taken into account when assessing this
result. The HER2 status of the CTCs did not impact on possible enrolment in the
trial. Thus, patients with HER2-negative CTCs who, as expected, did not benefit from
trastuzumab were also randomised. When planning future trials, the phenotype and genotype
characteristics of the detected cells should be included, if possible.
Clinical Applications of Liquid Biopsy in Metastatic Breast Cancer
Clinical Applications of Liquid Biopsy in Metastatic Breast Cancer
Improved prediction of prognosis
In the metastatic setting, CTC detection can complement the prediction of prognosis as well. In 2019, the course in 2436 patients
with metastatic disease was evaluated in a retrospective pooled analysis of 18 centres
[20] ([Table 2]). 54% of patients had already received systemic therapy for their metastatic disease.
CTC detection in all trials analysed was performed on the CellSearch system. However,
due to the higher concentration of CTCs in metastatic disease, a different cutoff
proved to be effective: in most trials 5 or more CTCs per 7.5 ml of blood was considered
elevated (or CTC-high). In the pooled analysis, patients with ≥ 5 CTCs per 7.5 ml
blood were classified as CTCaggressive and those with < 5 CTCs as CTCindolent. The analysis confirmed that the presence of increased CTC levels in metastatic disease
correlated significantly with shorter overall survival (median
OS: 36.3 in CTCaggressive vs. 16.0 months in CTCindolent, p < 0.0001) and this association was observed in all subtypes of the tumour [20]. In multivariate analysis, the following factors were associated with shorter OS:
prior treatment, poor differentiation, triple-negative phenotype, visceral metastasis,
and presence of ≥ 5 CTCs, with the CTC count as the strongest predictor of OS (HR
2.71, 95% CI 2.35 – 3.12, p < 0.0001).
Few trials explored the prognostic value of circulating DNA in metastatic disease ([Table 3]). Shaw et al. analysed blood samples from 112 patients with prior treatment [28]. Elevated cfDNA levels correlated with shorter overall survival. In addition, ctDNA
and CTCs were detected. ctDNA was identified by detecting mutations in the genes PIK3CA,
TP53, ESR1, and KRAS. Interestingly enough, elevated cfDNA levels were usually detected
in patients with high ctDNA levels. In terms of the mutation profile, the ctDNA reflected
the mutations that were detected in the CTCs.
Liquid biopsy treatment monitoring
According to several trials, clinical response in metastatic disease is reflected
by changes in the CTC numbers
[16]. A marked decrease in CTCs is usually noted even after the first cycle of palliative
chemotherapy. Smerage et al. and Martin et al. were able to demonstrate that after
the first cycle of therapy < 5 CTCs were detected in 47 – 57% of patients with initial
counts of ≥ 5 CTCs [51], [52]. Consistently high CTC numbers 3 – 4 weeks after the start of treatment on the other
hand indicate an increased risk of progression. This allows faster assessment of the
response than with conventional radiological diagnostic work-up. However, the clinical
consequences to be derived from consistently high CTC counts remain unclear. This
question was explored by the randomised phase-III trial of the US SWOG study group
[51]. A total of 595
patients with metastatic breast cancer received their first-line chemotherapy
in the trial. 43% of the 319 women with elevated CTC levels before treatment was initiated
continued to have ≥ 5 CTCs after the first cycle. These patients were randomised either
to a different treatment regimen or to continued treatment without change. The best
survival was observed in patients with low initial CTC levels (35 months), followed
by women whose high initial CTC levels dropped after the first cycle (23 months) and
patients with persistent CTC (13 months). Interestingly enough, since the change in
treatment did not yield the hoped-for improvement in prognosis, the CTC persistence
predicts resistance to traditional cytotoxic agents. It is possible that patients
with sustained high CTC levels could benefit from immunological, targeted or experimental
approaches.
The detection of circulating DNA was integrated into the translational adjunct programmes of several trials. In the
BEECH trial, Hrebien et al. addressed the question of how serial ctDNA measurements
could complement monitoring under chemotherapy [53]. In this randomized phase-II trial, patients with ER-positive, HER2-negative tumours
received first-line treatment with paclitaxel and the AKT inhibitor capivasertib versus
placebo. First, mutations in the baseline samples were evaluated, with the latter
then undergoing detection by mutation-specific ddPCR. As early as one week after the
start of treatment, changes in ctDNA levels were observed that could predict progression-free
survival. The best correlation with progression-free survival (PFS) was observed for
blood samples taken on day 1 of the second treatment cycle (q4w) (11.1 vs. 6.4 months,
hazard ratio 0.2). Both arms showed an equally strong drop in ctDNA levels, reflecting
the lack of clinical benefit of capivasertib that has since been demonstrated.
The PALOMA-3 trial also confirmed the high significance of early ctDNA testing [54]. In this phase-III trial, a total of 521 patients progressing under endocrine therapy
were randomised to treatment with fulvestrant and the CDK4/6 inhibitor palbociclib
vs. placebo. In 455 patients enrolled in the trial, the blood sample drawn before
starting treatment was analysed by multiplex dPCR for hotspot mutations in the PIK3CA
gene. At least one mutation was detected in 100 women, in 73 patients a blood sample
was also analysed on day 15 of treatment. In the palbociclib arm, the drop in ctDNA
between baseline and day 15 was markedly more pronounced than in the placebo arm.
Based on these results, it may be surmised that ctDNA dynamics may serve as a suitable
surrogate parameter for the early benefit assessment of new agents.
At the ASCO Symposium 2020 the results of two CDK4/6 trials were presented, which
had studied ctDNA measurements in their translational adjunct programmes [55], [56]. The PADA-1 trial evaluated the clinical significance of the ESR1 mutation detected
in cell-free DNA of 1017 patients under treatment with aromatase inhibitors and palbociclib
[56]. All patients presented with ER-positive, HER2-negative metastatic disease and received
first-line treatment in the trial. The serial blood samples were analysed by ddPCR.
Before treatment was initiated, the ESR1 mutation was detected in 3.2% of patients,
most of whom had already undergone adjuvant treatment with an aromatase inhibitor.
In 78% of the patients the mutation was eliminated from the cfDNA within the first
5 months of treatment. Patients with a mutation in the ESR1 gene before starting treatment
had a shorter PFS than women
without a mutation in the cfDNA. It remains to be seen how these results might
affect treatment decisions in ESR1mut patients in everyday clinical practice. In addition, ctDNA analysis was presented
at the ASCO Symposium 2020 in the context of the three trials on ribociclib, MONALEESA-2,
-3 and -7 [55]. The blood samples of 1503 patients in total were analysed by NGS for alterations
in 557 genes before endocrine-based treatment was initiated. Alterations in the genes
FRS2, PRKCA, MDM1, ERBB2, AKT1, and BRCA1/2 predicted a stronger PFS benefit for ribociclib
(statistical trend), while patients with alterations in genes CHD4, BCL11B, ATM, and
CDKN2A/2B/2C benefited little or not at all from ribociclib. Future trials must establish
whether the genes studied can contribute to the early detection of resistance in the
context of treatment monitoring.
Liquid biopsy-based therapeutic interventions
Today, treatment of the patient with metastatic disease is based on the predictive
properties of the primary tumour or metastases. This assumes the need for invasive
tissue sampling to obtain material for histological examination and may be associated
with complications in case of difficult locations. On the other hand, the clonal heterogeneity
of the malignant disease must be taken into account. Thus, individual metastases may
differ from each other in terms of phenotype and genotype, but different populations
can also be present within a metastatic mass. The analysed markers may also vary over
time. A meta-analysis of 39 trials demonstrated that 22.5% of patients with initially
ER-positive primary tumours developed ER-negative metastases [57]. Loss of HER2 status was observed in 21.3% of patients. In contrast, 9.5% of patients
with HER2-negative primary tumours developed HER2-positive metastasis. For this reason,
the Breast Committee of
the Working Group for Gynaecological Oncology (AGO-Kommission Mamma) recommends
a re-evaluation of the receptor status in metastatic disease.
Assuming that the blood-based biomarkers reflect the characteristics of the dominant
tumour populations, the possible use of the liquid biopsy as foundation for treatment
decisions is under intense discussion.
The STIC CTC study is one of the most important trials looking at CTC-based treatment choice
[58]. In this phase-III trial, a total of 778 women with hormone-receptor-positive HER2-negative
disease were randomised before starting their first-line therapy. In the CTC arm,
the choice of treatment was based solely on the results of the blood test: Patients
with < 5 CTCs received endocrine monotherapy, while women with ≥ 5 CTCs underwent
chemotherapy. Blood was also taken in the control arm and examined with the CellSearch
system, but the result remained blinded. In this arm, treatment was decided by the
attending physician based on the usual clinical criteria. As the study was initiated
in 2012, the study design did not include CDK4/6 inhibitors, which were only approved
later. The STIC CTC trial met its primary endpoint: the CTC-based choice of treatment
was not inferior to the physicianʼs decision, referred to as “treatment of
physicianʼs choice”. The clinical outcome (PFS and OS) was the same in both arms.
Interestingly enough, patients with discordant risk assessment (clinically low-risk
but with high CTC counts or clinically high-risk with low CTC counts) benefited from
chemotherapy in terms of overall survival. Since the STIC CTC trial did not include
any endocrine-based combined treatment with CDK4/6 inhibitors, the first-line therapy
selected most often at present in the hormone receptor-positive population, this trial
does not currently provide any recommendation for everyday clinical practice.
Unlike the STIC CTC trial, which analysed the CTC counts as the basis for treatment
decisions, the CirCe T-DM1 study explored the question of how the characteristics
of CTCs can affect the choice of treatment [59]. It studied whether patients with histologically HER2-negative disease (determined
for the primary tumour and/or metastases) and HER2-positive CTC status could benefit
from HER2-targeted treatment. A total of 154 women with prior treatment for breast
cancer were FisH-screened for HER2-amplified CTCs. In 14 cases at least one HER2-positive
CTC was detected, 11 of which were target-treated with T-DM1 in the trial. The response
rate was rather low at 9.1%. Only partial remission was achieved. The median PFS was
4.8 months and the OS 9.5 months. The lack of benefit of HER2-targeted therapy may
possibly be explained by the heterogeneous nature of the CTCs. The majority of CTCs
detected had a negative HER2 status and only 4.4% of CTCs
exhibited HER2 amplification.
Other CTC-based treatment concepts are currently being studied in the DETECT trials,
the worldʼs largest trial programme on CTC-based treatment interventions [50] ([Fig. 2]).
Fig. 2 Trial algorithm in the DETECT programme.
Unlike in diagnostic CTC work-up, ctDNA can already be considered in the routine clinical practice when choosing a particular
treatment. According to several trials, somatic genetic alterations in the PIK3CA,
ESR1, HER2, and PTEN genes can be detected and specifically targeted as “targetable
mutations” ([Table 7]).
Table 7 Possible alterations detectable in the ctDNA and their potential significance.
|
Gene/Marker
|
potential clinical significance
|
|
ESR1
|
Imparting endocrine resistance and unfavourable prognosis, depending on the variant
resistance to everolimus (ESR1D538G and Y537S), exemestan (D538G, ESR1mut), fulvestrant (Y537S, Y537C), relative fulvestrant sensitivity (ESR1mut)
|
|
PIK3CA
|
Prediction of response to therapy with PI3K inhibitor (alpelisib approved in Europe
in 2020); potentially useful in treatment monitoring
|
|
AKT1
|
Prediction of response to AKT inhibitors such as capivasertib (AKT1E17K)
|
|
HER2
|
Prediction of the response to neratinib in HER2mut
Treatment monitoring under anti-HER2 therapy
|
|
TP53
|
Imparting resistance to treatment
|
|
PTEN
|
Imparting resistance to treatment
|
|
BRCA
|
Prediction of the response to PARP inhibitors and platinum salts in somatic BRCA mutations
in the ctDNA
|
|
Microsatellite instability (MSI), loss of heterozygosity (LOH)
|
Prediction of the response to targeted and immunological therapeutic approaches
|
The first agent with liquid biopsy-based indication was approved as a result of the
SOLAR 1 trial [30]. Here, the PI3K inhibitor alpelisib in combination with fulvestrant was studied
in patients with hormone receptor-positive HER2-negative disease. Alpelisib inhibits
the enzyme PI3 kinase coded by the PIK3CA gene. Mutations in the PIK3CA gene are seen
in up to 40% of patients in advanced stages and result in increasingly aggressive
disease by activating the PI3K/Akt/mTOR signaling cascade. In the SOLAR 1 trial, a
total of 572 patients were randomised to treatment with fulvestrant and alpelisib
versus fulvestrant and placebo. The patients suffered from metastatic disease and
had already undergone prior endocrine treatment. In all women, the PIK3CA mutation
status was determined in the tumour tissue and in some cases also in the ctDNA. Detection
of a mutation was associated with a significant PFS benefit when alpelisib was added,
regardless
of whether the mutation was detected in the tissue or in the ctDNA. Alpelisib
has already been approved by the U. S. FDA in 2019 and by the European Medicines Agency
in 2020.
With modern sequencing techniques, it is possible to study several potentially treatment-relevant
mutations simultaneously. The PlasmaMatch trial [60] followed this approach, in which blood samples from a total of 1044 women with metastatic
breast cancer were screened. A number of genetic alterations in the ctDNA were detected.
The TP53 gene was most often affected (44.1%), followed by PIK3CA (34.9%), ESR1 (oestrogen
receptor 1) (33.1%), PTEN (6.9%), HER2 (6.4%), and AKT1 (5.0%). The study design provided
for mutation-triggered targeted treatment. For example, patients with ESR1 gene mutations
were treated with fulvestrant, while women with HER2 gene mutations received anti-HER2
treatment with neratinib. In case of mutations in the PTEN or AKT1 gene, treatment
with capivasertib was initiated. After analysis of the response, treatment with neratinib
and capivasertib proved particularly promising (response rates: neratinib 25%, capivasertib
22 – 33%).
[Table 8] summarises the current knowledge on liquid biopsy in early and advanced breast cancer.
Table 8 Clinical significance of CTCs and circulating DNA in breast cancer.
|
|
CTCs
|
Circulating DNA
|
|
M0
|
Prognostic significance
|
Very high (confirmed in meta-analyses)
|
Probably high; limitation: small case numbers, rather brief follow-up, differences
in methodology
|
|
Treatment monitoring
|
Persistence after (neo)adjuvant chemotherapy predicts worse outcome
|
Persistence after (neo)adjuvant chemotherapy predicts worse outcome; ctDNA dynamics
correlate with pCR
|
|
Follow-up complement
|
Potentially significant, especially in HR positive disease: CTC detection within 5
years post-diagnosis predicts an increased risk of relapse
|
Potentially significant: ctDNA positivity predicts an increased risk of relapse
|
|
Liquid-biopsy based therapeutic interventions
|
No positive trials to date, potential unclear; TREAT CTC trial without benefit of
trastuzumab in persistent CTC (HER2 status of CTCs not considered)
|
No positive trials to date, potential unclear
|
|
M1
|
prognostic significance
|
Very high (confirmed in meta-analyses)
|
Probably high; limitation: few trials, small case numbers, differences in methodology
|
|
Treatment monitoring
|
High potential: Persistence after 1st cycle chemotherapy correlates with response,
clinical consequence unclear
|
High potential: Persistence after 1st cycle of treatment correlates with response,
clinical consequence unclear
|
|
Liquid biopsy-based therapeutic interventions
|
First positive trial (STIC CTC): CTC-based choice of first-line therapy is not inferior
to oncologistʼs choice; clinical consequence unclear; further trials pending (e.g.
DETECT study programme)
|
Main area of use: Detection of somatic mutations in the ctDNA as basis of indication for targeted therapy:
Alpelisib approved in the USA and Europe for patients with a mutation in the PIK3CA
gene
|
Summary
-
Liquid biopsy refers to the study of circulating tumor cells and nucleic acids (DNA/RNA)
in the blood.
-
The prognostic significance of CTC detection in patients with breast cancer is very
high in both early and metastatic stages.
-
The dynamics of CTCs and ctDNA correlate with response to palliative treatment.
-
Oncologic research currently focuses on liquid biopsy-based therapeutic interventions
in metastatic breast cancer. The PI3K inhibitor alpelisib is the first agent approved
in this context.
