Blood-based biomarkers in breast cancer: From proteins to circulating tumor cells to circulating tumor DNA

Abstract

Biomarkers are the key to personalized treatment in patients with breast cancer. While tissue biomarkers are most useful in determining prognosis and upfront predicting response to therapy, circulating protein biomarkers such as CA 15-3 and carcinoembryonic antigen are mainly used in monitoring response to endocrine or chemotherapy in patients with advanced disease. Although several centers measure biomarkers in asymptomatic patients following curative surgery for primary breast cancer, the clinical utility of this practice is unclear. Promising new biomarkers for breast cancer include circulating tumor DNA and circulating tumor cells. In contrast to circulating protein biomarkers, measurement of circulating tumor DNA–based biomarkers is potentially useful in identifying mechanisms of resistance to ongoing therapies as well as identifying new targets for further treatment. To increase clinical utility, both the established and emerging circulating biomarkers should where possible be incorporated into randomized trials evaluating new therapies in patients with breast cancer.

Keywords

Breast cancer biomarkers circulating CA 15-3 circulating tumor DNA circulating tumor cells

Measurement of specific biomarkers is mandatory for the optimum management of patients with invasive breast cancer. Thus, tissue-based biomarkers such as estrogen receptors (ERs), HER2, Ki67 and multigene signatures are used for determining prognosis and guiding systemic therapy. Blood-based biomarkers such as CA 15-3 and carcinoembryonic antigen (CEA), however, tend to be used in postoperative surveillance following primary treatment and in monitoring therapy in advanced disease. Tissue-based biomarkers have been extensively reviewed in recent years.^1–3 In contrast, there have been few recent comprehensive reviews on blood-based biomarkers, despite the emergence of promising new blood-based biomarkers such as circulating tumor DNA (ctDNA) and circulating tumor cells (CTC; Table 1). The aim of this article is therefore to discuss the clinical utility of established serum biomarkers and potential clinical utility of the emerging blood-based biomarkers (ctDNA and CTC). As CA 15-3 is the most widely investigated of the circulating biomarkers in breast cancer, most of the article will focus on it.

Table 1.

Main type of circulating biomarkers in breast cancer, together with their main uses/potential uses, advantages, and disadvantages.

Type of biomarker	Example	Key uses/potential uses	Advantages	Disadvantages
Protein	CA 15-3, CEA, TPA, TPS, sHER2	Surveillance, therapy monitoring, and prognosis	Easy and cheap to measure, automated assays available	Lack sensitivity and specificity
ctDNA	ESR1, PIK3CA, TP53	Identification of therapy resistance mechanism, and monitoring therapy	High specificity for malignancy, only non-invasive way to identify mechanism of therapy resistance	Expensive, assays not widely available, and labor intensive
CTC		Prognosis and monitoring therapy	Allow measurement of biomarkers at DNA, mRNA, and protein levels	Expensive, not widely available, and labor intensive

TPA: tissue polypeptide antigen; TPS: tissue polypeptide–specific antigen; sHER2: soluble HER2; ctDNA: circulating tumor DNA; CTC: circulating tumor cells.

Traditional protein biomarkers

Use in postoperative follow-up following primary treatment for breast cancer

Following curative intent surgery (and adjuvant treatment) for several different types of primary cancer, a common practice is to follow-up patients at regular intervals, at least for the first 5 years. This practice is based on the widely held assumption that the early detection of recurrent/metastatic disease and initiation of therapy enhances the chance of cure or results in improved survival. During this follow-up period, serial levels of biomarkers may be measured. Thus, after curative surgery for colorectal cancer, measurement of serial levels of CEA is widely recommended,^4–7 as this practice has been shown to result in improved patient outcome.^8–11 In contrast, a prospective randomized trial concluded that measurement of CA 125 levels did not lead to an improved outcome for patients in remission following first-line therapy for ovarian cancer.¹² Although most expert panels are opposed to the measurement of serum biomarkers in the follow-up of asymptomatic women following primary surgery and adjuvant therapy for breast cancers,^13–16 several centers continue to test for these biomarkers.^17–19 The biomarkers most frequently measured in this setting are CA 15-3, CEA, tissue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS) and the soluble form of HER2 (sHER2).^20–24

A pooled analysis of early studies involving the postoperative measurement of CA 15-3 concluded that 67% of 352 asymptomatic patients had increased CA 15-3 concentrations either before or at the time of recurrence.²⁰ In 1320 patients lacking evidence of recurrence at the time of reporting, 92% had CA 15-3 concentrations within the reference ranges. The lead times from concentration of biomarker increasing to clinical diagnosis of recurrence varied from 2 to 9 months.²⁰

More recent studies measuring multiple biomarkers reported increased accuracy in detecting early recurrences.^21–24 Thus, by combining measurement of both CA 15-3 (with a cut-off value of >60 kU/L) and CEA (with a cut-off value of >10 µg/L) and excluding patients with locoregional recurrences, Molina et al.²¹ reported a sensitivity of 64% and specificity of 99% for detecting recurrent disease. Based on a confirmed 100% increase in biomarker levels, Stieber et al.²² reported that combined measurement of CA 15-3 and CEA detected early metastasis with a sensitivity of 66.3% and a specificity of >98%. However, using specific cut-off values (30 kU/L for CA 15-3 and 4 µg/L for CEA), specificity was found to be 86.3% and sensitivity 70.6%.²² Sensitivity, however, could be further enhanced by measuring additional biomarkers such as CA 125, sHER2, and CYFRA 21-1.²³ In another study that measured multiple biomarkers, Nicolini et al.,²⁴ using individualized cut-off values, found that combined measurement of CA 15-3, CEA and TPA detected early recurrence with a sensitivity of 93% and a specificity of 97.6%.

The efficiency of biomarkers for detecting early recurrence depends on several different factors. First, it depends on how an increase in biomarker concentration is defined. As is clear from above, in some studies, a particular increase above a specific threshold was used, while in others, a specific percentage increase compared to the previous level was used. To date, an optimized or validated definition of a clinically significant biomarker level increase has not been established for any serum biomarker. According to Soletormos et al.,²⁵ the critical difference between successive biomarker measurements should be based on both the analytical imprecision of the assay (CV_a) and the normal intra-individual biological variation (CV_i). Based on CV_a values of 11.2% for CA 15-3, 9.5% for CEA, and 11% for TPA, these authors calculated that successive biomarker levels should differ by 30%, 31%, and 72%, respectively, for their p values to be significant at a 0.05 level.²⁵ Assuming these results can be confirmed, different biomarkers would require different percentage increases in sequential values to be clinically relevant.

The efficiency of biomarkers to detect early recurrences also depends on the site(s) of recurrence, being most sensitive for detecting distant metastases and of little value in diagnosing locoregional recurrences.^26–29 Highest detection rates are found in patients with bone or liver metastases. In contrast, biomarkers are rarely increased in patients with locoregional recurrences or lung-only metastasis.^26–29 Other factors likely to affect the ability of biomarkers to detect early recurrences include frequency of measurement, aggressiveness of the primary cancer, specific biomarker or biomarker panel utilized, ER status of primary cancer,²¹ and imaging method used to detect recurrent disease.

Although measurement of biomarkers can result in the detection of occult recurrences, most expert panels recommend against their testing in asymptomatic women during follow-up after primary treatment.^13–16 This recommendation is based on the lack of high-level evidence demonstrating that an intensive follow-up strategy improves outcome or enhances quality of life compared to a minimal follow-up strategy. This lack of evidence is primarily based on findings from two old Italian randomized prospective trials that began in the 1980s and were published in the early 1990s.^30,31 Both these studies concluded that follow-up with regular physical examinations and annual mammography was as effective as a more intensive strategy that also involved regular imaging and standard laboratory testing, with respect to overall survival (OS).

As these trials commenced almost 30 years ago, their relevance to the modern management of patients with breast cancer is questionable.³² In particular, these early trials failed to include measurement of any of the above-mentioned cancer-associated biomarkers. Furthermore, compared to modern imaging instrumentation, the older methods were less accurate in detecting early recurrences. Perhaps, the biggest transformation in breast cancer patient management since these trials were performed, however, is the multiplicity of new systemic therapies currently available for patients with recurrent breast cancer, especially for ER-positive and HER2-positive cancers.^33–38

Thus, when these old Italian trials were ongoing, tamoxifen was the only form of systemic endocrine therapy available. In the meantime, additional forms of endocrine therapy such as the aromatase inhibitors and the selective estrogen receptor downregulator (SERD), fulvestrant have become available.³⁴ In addition, these endocrine agents can be combined with cyclin-dependent kinase (CDK) 4/6 inhibitors³⁷ or the mechanistic target of rapamycin (mTOR) inhibitor, everolimus to further enhance outcome.³⁸ Although resistance may develop to one member of a class of endocrine therapy (e.g. tamoxifen), tumor regression may occur with a different class compound.^33,34

Another major difference in systemic treatment for recurrent breast cancer between 25 years ago and the present is the current availability of multiple agents for targeting HER2-positive tumors.^35,36 Prior to the approval of trastuzumab, the outcome for patients with HER2-positive breast cancer was considerably worse than that for HER2-negative patients. However, with the availability of anti-HER2 therapies, especially trastuzumab, the prognosis for HER2-positive patients has greatly improved. Furthermore, HER2-positive patients who become resistant to trastuzumab can be further treated with other forms of anti-HER therapy such as trastuzumab plus pertuzumab, lapatinib plus chemotherapy, or trastuzumab emtansine (T-DM1).^35,36

Although the above two Italian trials failed to measure cancer biomarkers, a subsequent prospective study included CA 15-3 as part of the follow-up strategy.³⁹ In this trial, 472 patients with newly diagnosed breast cancer diagnosed between 1991 and 1995 were randomized to 3 versus 6 monthly follow-up and to standard investigations (full blood count, standard biochemical testing, and CA15-3 measurement at every visit) versus no routine testing. Following a median follow-up time of 4.2 years, there was no significant difference in either disease-free or OS between those with three versus six monthly follow-up or between those with routine versus no routine diagnostic testing. The mean cost of follow-up per patient varied from €1050 (6 monthly and no routine tests) to €2269 per follow-up (3 monthly with routine tests) and the mean cost per detected recurrence varied from €4166 (6 monthly and no routine tests) to €9149 Euro (3 monthly with routine tests). Based on these results, the authors concluded that follow-up examination every 6 months was as effective as every 3 months in detecting recurrence. However, the less frequent follow-up schedule was cost saving. The authors also concluded that routine blood tests and imaging were unnecessary in the follow-up of an asymptomatic breast cancer patient but doubled the costs of follow-up. With the multiplicity of arms in this trial, the relatively short follow-up period of 4.2 years and the modest number of patients investigated (i.e. 472), it is unclear whether this trial had sufficient power to address whether intensive follow-up with CA 15-3 testing resulted in a superior outcome to that of a minimalist follow-up strategy.

In the absence of a modern and properly designed prospective randomized clinical trial, there is inadequate evidence to recommend for or against the use of biomarkers in the surveillance of patients following a diagnosis of breast cancer. In such a situation, it might be appropriate to implement the practice of informed consent, that is, patients are informed of the advantages and limitation of the relevant testing. With such a strategy, the ultimate decision as to whether or not to have testing carried out is then made by the patient in consultation with their doctor.⁴⁰

In this situation, the doctor should discuss some or all the following with their patients:

Measurement of serial levels of biomarkers may lead to the early detection of recurrent disease. However, there is no evidence that the use of biomarkers to detect early recurrences results in better outcome. There is, however, also no evidence that measurement does not result in a better outcome, that is, the absence of evidence due to appropriate trials not having been performed is not evidence of the absence of a potential benefit.

Use of biomarkers may give false findings that could lead to unnecessary investigations and unnecessary treatments. These unnecessary investigations may result in serious side-effects and be costly.

The optimum cut-off points for defining biomarker increases are unknown.

Recurrent disease can occur without increased biomarker levels.

Anticipation of biomarker results can lead to anxiety and stress.

Measurement of biomarkers results in increased costs.

Finally, from a biological perspective, early administration of treatment to a patient with an emerging recurrence might be expected to result in better outcome than administration when the recurrence is clinically evident. However, this remains to be shown in a randomized clinical trial involving one or more of the above-mentioned biomarkers, use of modern imaging methods, and administration of modern treatments.

Clearly, with such uncertainty regarding the possible value of measuring biomarkers in asymptomatic women following primary treatment, a prospective randomized trial is necessary to evaluate the potential benefit of serial biomarker testing on patient outcome or quality of life. Although designs for such a clinical trial have previously been proposed,^41,42 to the authors knowledge, none have started. Until such a clinical trial is carried out, it is likely that the disconnection between guideline recommendation and the clinical practice of some centers measuring biomarker in asymptomatic breast cancer patients will continue.

Use in monitoring response to chemotherapy in advanced disease

In general, changes in serial levels of biomarkers, especially CA 15-3 and CEA, correlate with response to chemotherapy in patients with advanced breast cancer.^43–51 Indeed, the use of biomarkers to monitor therapy has several advantages over conventional criteria (e.g. International Union against Cancer criteria) including lower costs, more objective measurement, and more convenience for patients.

While most expert panels are opposed to the use of biomarkers in the follow-up of asymptomatic patients following primary treatment for breast cancer, they cautiously recommend use of biomarkers as an aid in monitoring treatment, especially in patients with non-evaluable disease, that is, in patients whose response that cannot be readily determined using standard imaging.^13–16 Approximately, 10%–40% of patients with breast cancer have non-assessable disease, such as those with irradiated lesions, pleural effusion, ascites, lytic bone disease, and sclerotic bone disease.⁵¹ However, for patients who have assessable disease, changes in CA 15-3 concentrations should not be used alone in monitoring response to chemotherapy.

Although in most situations, biomarkers tend to increase with disease progression and decrease with regression, paradoxical increases, known as spikes or surges, can occur after the commencement of chemotherapy, especially in patients with extensive metastatic burden.^48,52,53 These transient increases are usually not related to tumor progression but appear to be result from therapy-mediated apoptosis or necrosis of tumor cells. However, in one study, patients displaying a surge were found to have a significantly higher risk of disease progression than patients without a surge.⁵³

As with biomarkers during postoperative surveillance, the optimum frequency for measurement when monitoring treatment in advanced breast cancer is unclear. According to the European Group on Tumor Markers (EGTM) panel, biomarkers should be determined before every course of chemotherapy and at 3-month intervals for patients receiving hormone therapy.⁵⁴ In contrast, a joint European School of Oncology and European Society of Medical Oncology panel stated that evaluation of response to therapy should generally occur every 2–4 months following endocrine therapy or after two to four cycles for chemotherapy, depending on the aggressiveness of the disease, its location, and extent of metastatic involvement.⁵⁵

Similar to the situation in postoperative surveillance, validated definitions of biomarker increase to predict disease progression and decreases to reflect disease regression have not been established. However, according to the EGTM, a clinically significant increase in biomarker concentration occurs if there is an increase of at least 25% over the previous value. It was recommended that the increased concentration should be confirmed with a second sample taken within 1 month. The Panel also stated that a confirmed decrease in biomarker concentration of >50% was consistent with tumor regression.⁵⁴

In addition to chemotherapy and endocrine therapy, several other forms of systemic therapy are currently available for patients with recurrent or advanced breast cancer. These include anti-HER2 therapies, CDK4/6 inhibitors, and mTOR inhibitors (see above). Although biomarkers such as CA 15-3 and CEA have been widely investigated for monitoring response to chemotherapy or endocrine therapy, their utility in evaluating response to the newer forms of therapy is less clear. However, CA 15-3 may not be a reliable biomarker in monitoring response to the mTOR inhibitor, everolimus, as this compound has been reported to increase its levels in a patient who was apparently responding to the drug.⁵⁶ This paradoxical increase may relate to everolimus-induced interstitial lung disease (ILD).⁵⁶ If CA 15-3 levels increase while receiving everolimus, further investigations, such as high-resolution chest CT and pulmonary function tests, should be carried out to establish the presence of ILD.⁵⁷

Use in monitoring response to anti-HER2 therapy

While neither CA 15-3 nor CEA have been widely investigated for monitoring response to anti-HER2 therapy, several reports have evaluated sHER2 (extracellular domain of HER2 which is also known as p95) in this situation. sHER2 might be expected to be a good biomarker for monitoring response to anti-HER2 treatments as these therapies are only administered to patients whose tumor exhibits HER2 gene amplification or overexpression. Indeed, multiple studies have evaluated the potential utility of sHER2 for monitoring response to drugs such as trastuzumab and lapatinib.^35,58 Based on a pooled analysis of data from seven trials that investigated a potential value for sHER2 in evaluating response to trastuzumab-based therapy in patients with advanced breast cancer, Ali et al.⁵⁹ reported that 191/307 (62%) of patients exhibited a significant decrease (>20%) in sHER2 concentrations, while 116 (38%) failed to show a decline. Objective response rate was found in 57% of the patients who achieved a decline in serum sHER2, in contrast to only 28% for patients who did not. Patients showing a decrease also had a significantly longer time to disease progression, longer duration of response, and longer OS than those without a decrease. Similarly, in patients treated with lapatinib, those with a ≥20% decrease in sHER2 levels exhibited a significantly increased progression-free survival (PFS) and OS compared to patients not displaying such a decrease. Conversely, those with a ≥20% increase from baseline had a significantly lower overall response rate (ORR) and shorter PFS.⁶⁰

While overall, patients with deceasing levels of sHER2 appeared to do better when treated with anti-HER2 therapy than those whose levels increased, the available data suggests that this biomarker has limited value for monitoring individual patients. Because of this lack of consistency, measurement of sHER2 is not currently recommended for monitoring response to anti-HER2 or indeed any other therapy in patients with breast cancer.

DNA-based biomarkers

While research on new circulating protein biomarkers for breast cancer has waned in recent years, there has been a major upsurge of work on DNA-based biomarkers. Indeed, in recent years, much of the research on blood-biomarkers in breast cancer has shifted from protein-based to DNA-based molecules. DNA, like proteins, is released from tumors into the circulation. Compared to tissue biopsies, use of ctDNA allows minimally invasive access and ease of serial monitoring. Depending on factors such as specific treatment administered, tumor load, tumor location, tumor cell turnover, extent of tumor necrosis, and access to the vasculature, ctDNA can vary from <0.01% to >90% of the total DNA in blood.⁶¹ As with the protein biomarkers, the extent of elevations in ctDNA levels and number of patients having increased levels is greater in women with advanced than in those with localized disease.⁶¹

Compared to circulating protein biomarkers, use of ctDNA has several advantages including greater dynamic range and shorter half-life (<2.5 h).⁶¹ These two characteristics should enable ctDNA to be a more sensitive biomarker for monitoring tumor progression and evaluating response to therapy. This short half-life, however, requires careful timing in specimen collection. Another advantage of ctDNA-based biomarkers is that they should be more specific than traditional biomarkers for malignancy, thus decreasing false-positive rates. Perhaps, the main advantage of ctDNA over the traditional biomarkers, however, is that its measurement may be able to identify the genetic alterations responsible for acquired resistance to specific therapies. Importantly, this capability can potentially identify new treatment options for patients with a progressing tumor (see below).

Several proof-of-concept studies have been carried out aimed at evaluating a potential clinical utility for ctDNA in patients with breast cancer. Preliminary results suggest that ctDNA can preclinically signal the emergence of early recurrence in asymptomatic patients who have undergone curative surgery for primary breast cancer, identify mechanisms of acquired resistance to ongoing treatments, and monitor response to therapy in patients with metastatic disease.

Use in postoperative follow-up following primary treatment for breast cancer

In one of the first studies to investigate ctDNA in early breast cancer, Garcia-Murillas et al.,⁶² using an assay based on the primary tumor genetic alterations, tracked plasma mutations in 55 patients who had received neoadjuvant chemotherapy. Of the 15 patients who subsequently relapsed, 12 (80%) had ctDNA detected during follow-up. Of the patients who remained free of recurrence, 24/25 (96%) were negative for ctDNA. Measurement of serial samples of ctDNA was found to signal disease relapse, with a median lead time of 7.9 months over clinical relapse. In another small study (n = 20 patients), Olsson et al.⁶³ reported that the presence of ctDNA preceded clinical diagnosis of recurrence in 12/14 (86%) patients with an average lead time of 11 months. Six patients without evidence of recurrence had no detectable ctDNA. A limitation of both these studies was that ctDNA levels were not compared with conventional breast cancer biomarkers such as CA 15-3 or CEA. It is thus not possible to conclude if ctDNA has superior accuracy than the currently available protein biomarkers for detecting early recurrence in patients with breast cancer.

Use in identifying mechanism of resistance and new therapeutic targets

Although traditional biomarkers tend to increase with the emergence of therapy resistance and the development of recurrent disease (see above), these biomarkers are unable to identify mechanisms of therapy resistance. As mentioned above, measurement of mutations in ctDNA, however, can potentially identify mechanisms of resistance. This in turn may result in the identification of new targets for further therapy.

ER which is universally used for predicting benefit from endocrine therapy in breast cancer is mutated in almost half of patients with metastatic disease.^64,65 Such mutations are mostly detected in women who have been receiving aromatase for metastatic breast cancer and are mostly present in the ligand-binding domain of ER. Preclinical studies showed that presence of specific mutations (e.g. Y537 S and D538G) leads to constitutive activation of ER (i.e. activation in the absence of estradiol), resistant to aromatase inhibitors, and decreased sensitivity to tamoxifen and to the selective ER degrader (selective estrogen receptor modulator (SERM)) compound, fulvestrant.^64,65

Several reports have recently shown that ESR1 (gene encoding ER) mutations can be detected in plasma.^66,67 Thus, in a recent prospective trial, ESR1 mutations were detected in 22/39 (56%) of metastatic breast cancer patients who developed progressive disease while receiving aromatase inhibitors.⁶⁸ Indeed, these mutations were present a median of 6.7 months prior to clinical evidence of progression. In addition to ESR1 mutations, mutations in RAS were found in 6/39 (15%) of patients with progressive disease. It is, however, unclear if mutant RAS contributed to aromatase inhibitor resistance or cancer progression.

Consistent with the preclinical data, preliminary clinical findings suggest that the emergence of specific mutations in the ESR1 gene may be responsible for acquired resistance to aromatase inhibitors.^66,69 Thus, preliminary data showed that the presence of plasma ESR1 mutations was associated with shorter progression-free interval on subsequent aromatase therapy compared to patients with wild-type ESR1.⁶⁶ Furthermore, patients with ER-positive advanced breast cancer with ctDNA-based ESR1 mutations exhibited improved PFS when treated with fulvestrant compared with the aromatase inhibitor, exemestane.⁶⁹ In contrast, patients with ctDNA wild-type ESR1 had similar PFS irrespective of the type of endocrine treatment administered.

Provided this preliminary finding can be confirmed, ER-positive advanced breast cancer patients who develop resistance to an aromatase inhibitor as a result of ESR1 mutations might be administered fulvestrant rather than aromatase inhibitors. Indeed, several new drugs that are active against mutant ER are currently in development.^70–72 In contrast to fulvestrant, some of these compounds can be administered orally.

Another potential ctDNA-based biomarker is HER2 gene amplification. Like ER, measurement of HER2 gene amplification/overexpression is universally measured in breast cancers, its main use being for predicting benefit from anti-HER2 therapy such as trastuzumab (Herceptin), lapatinib, pertuzumab, or trastuzumab emtansine (T-DM1).⁷³ Recently, HER2 gene amplification was detected in plasma during the follow-up of breast cancer patients.⁷⁴ Whether such detection in patients with previous HER2-negative cancers can be used to predict response to anti-HER2 therapy remains to be shown.

A further ctDNA-based biomarker with potential therapeutic relevance is the mutational status of PIK3CA. PI3K is involved in downstream signaling from several clinically relevant membrane receptors such as HER2 and epidermal growth factor receptor (EGFR). Currently, multiple trials are ongoing evaluating PI3K inhibitors in different cancers including breast cancer. In a phase III randomized clinical trial in patients with advanced breast cancer, combined treatment with the PI3K inhibitor, buparlisib and fulvestrant, resulted in increased PFS and OS compared to fulvestrant alone in patients with ctDNA PIK3CA mutations but not in those with ctDNA wild-type PIK3CA.⁷⁵ Provided these results can be confirmed, the mutational status of PIK3CA might be useful in identifying patients who are likely to benefit from combined treatment with buparlisib and fulvestrant.

Use in monitoring therapy in advanced disease

In one of the few studies to have evaluated the potential value of ctDNA in monitoring response to treatment in advanced breast cancer, Dawson et al.⁷⁶ compared ctDNA with CA 15-3 and CTC in 30 patients receiving chemotherapy. The ctDNA-based biomarkers measured were mainly derived from two genes previously identified to be mutated in the patients’ tumors, that is, PIK3CA and TP53 (encodes p53). Although 52 patients were recruited, only 30 had identifiable tumor-related ctDNA alterations suitable for monitoring. Of these 30, 29 (97%) had detectable alterations in the circulation. In contrast, CTC were found in 26/30 (87%) and elevated concentrations of CA 15-3 in 21/27 (78%) of the patients investigated. ctDNA and number of CTC but not CA 15-3 levels were associated with adverse prognosis. Although this was a preliminary report, the authors⁷⁶ concluded that measurement of ctDNA provided a greater dynamic range, better correlation with alterations in tumor load and an earlier signal of response to treatment than CTC or CA 15-3.

CTC

Malignant cells, similar to circulating protein biomarkers and ctDNA, are shed into the circulation from different types of solid cancers. Such cells are referred to as CTC. CTC can exist as individual cells or form clusters. Although less common, clusters of CTC have been reported to be more efficient in forming metastasis. However, the proportion that eventually produces metastatic lesions is thought to be relatively small. As CTC constitute a low proportion of the total number of cells present in blood from patients with cancer (1–10 cells per one million nucleated cells and one billion red cells or 10 mL of blood), their detection first requires an enrichment step.

Although several different methods have been described for isolating and measuring CTC,^77,78 the most widely investigated is the CellSearch Circulating Tumor Cell kit (Menarini Silicon Biosystems Inc).^79–81 With this system, CTC are initially enriched by binding to epithelial cell adhesion protein (EpCAM) which is assumed to be present only on epithelial-derived cells. Further selection of CTC is based on cells being cytokeratin-positive (CK8, 18, and 19-positive), CD45-negative, and presence of a nucleus. A limitation of the CellSearch method is that some CTC may undergo epithelial–mesenchymal (EM) transition. As a result, EpCAM is lost; the consequence being that such cells cannot be detected with this system. Potential markers for identifying mesenchymal cells include plastin-3 and vimentin.^82,83

Use in determining prognosis and monitoring response to therapy

Several studies have shown that high levels of CTC (e.g. ≥5 CTC per 7.5 mL of blood) is an independent predictor of adverse prognosis in patients with either early or advanced breast cancer.^80,81,84,85 To evaluate whether measurement of CTC has potential clinical utility, that is, can alter patient management, Smerage et al.⁸⁶ randomized patients with advanced breast cancer containing high levels of CTC following a single cycle of adjuvant chemotherapy, to continue the same treatment or to switch to a different treatment. However, no significant difference in median OS was observed between the two groups. Thus, for patients with persistently increased CTCs after first-line chemotherapy, early changing to an alternate therapy was not found to be effective in prolonging OS.

In an attempt to enhance the clinical use of CTC, several studies are currently focusing on the molecular characterization of these cells. Thus, several reports have compared the ER/progesterone receptor (PR) status of CTC vis-à-vis primary breast tumors. Overall, concordance between the two locations ranged from 40% to 70%.⁸⁷ To the authors’ knowledge, no study has yet evaluated a potential value for measurement of ER/PR in CTC. As in plasma, ESR1 mutations have also been detected in CTC,⁸⁸ but it is unclear how such mutations related to those in tumor tissue or ctDNA.

Similarly, although HER2 has been detected in CTC, it remains to be shown whether the oncoprotein has predictive value for anti-HER2 therapy in this setting. The HER2 status of CTC, however, is not fixed as HER2-positive and HER2-negative cells have been shown to interconvert spontaneously. This switching could potentially lead to an erroneous interpretation of the HER2 status of CTC. Two prospective trials, however, are currently addressing if the presence of HER2-posive CTC in patients negative for tissue HER2 can predict response to anti-HER2 therapy (NCT01619111 and NCT01975142).

Conclusion

Currently, the best-established role for serum/plasma biomarkers in breast cancer is as an aid in monitoring response to cytotoxic chemotherapy in patients with advanced disease. Although no study appears to have directly compared the clinical utility of the available biomarkers for this purpose, CA 15-3 and CEA are the most widely used. With the exception of patients who have non-evaluable disease, these biomarkers, however, should not be used alone in evaluating response to therapy. Although used in some centers, the utility of biomarkers in detecting occult disease in asymptomatic patients following primary treatment is not supported by most expert panels. Hopefully, this issue can be addressed in a randomized clinical trial in the future. Such a trial, however, would be logistically difficult to perform, take a considerable amount of time and be expensive to undertake.

Promising new circulating biomarkers in breast cancer include CTC and ctDNA. ctDNA in contrast to the traditional protein biomarkers has the potential to identify emerging mechanisms of therapy resistance as well as new targets for treatment. To accelerate progress in this area, ctDNA-based biomarkers should be incorporated into new therapeutic trials being performed in patients with breast cancer. The availability of validated ctDNA biomarkers should be another step along the road to personalized treatment for patients with breast cancer.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors wish to thank Science Foundation Ireland, Strategic Research Cluster Award (08/SRC/B1410) to Molecular Therapeutics for Cancer Ireland (MTCI), the Cancer Clinical Research Trust, and the BREAST-PREDICT (CCRC13GAL) program of the Irish Cancer Society for funding this work. The funding organizations had no input into the writing of this manuscript.

ORCID ID

Michael J Duffy

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