Circulating Tumour Cells in Clinical Practice: Can Lessons Learned From Locally Advanced/Metastatic Breast and Prostate Cancers Guide Their use for other Cancer Types? Lights and Shadows

Michela Buglione, Luca Triggiani, Nadia Pasinetti, Loredana Costa, Sara Pedretti, Marta Maddalo, Paolo Borghetti, Camillo Almici, Salvatore Grisanti, Stefano Maria Magrini

Michela Buglione, Luca Triggiani, Nadia Pasinetti, Loredana Costa, Sara Pedretti, Marta Maddalo, Paolo Borghetti, Stefano Maria Magrini, Radiation Oncology Department, University and Spedali Civili, Brescia; P.le Spedali Civili 1, Brescia, Italy
Camillo Almici, Immunohematology Department, Spedali Civili, Brescia; P.le Spedali Civili 1 – Brescia, Italy
Salvatore Grisanti, Medical Oncology Department, University and Spedali Civili, Brescia; P.le Spedali Civili 1, Brescia, Italy.

Correspondence to: Michela Buglione, Radiation Oncology Department, University and Spedali Civili, Brescia, Piazzale Spedali Civili, 1, Italy.
Email: michela.buglione@unibs.it
Telephone: +39-030-3995271
Fax: +39-030-399601
Received: July 11, 2015
Revised: September 25, 2015
Accepted: September 30, 2015
Published online: December 10, 2015


Circulating tumour cells are cells probably involved in different phases of the metastatic process. They can be identified using standardized and repeatable methods and are characterized by EpCAM; Citokeratin 8, 18, 19 and DAPI positivity and CD45 negativity. CTC’s identification in patients with metastatic breast and prostate cancer has been demonstrated to have a prognostic and predictive role. It is not still clear if these cells can be used also in patients with non metastatic breast and prostate cancer, or in patients with different diseases (eg. metastatic and/or locally advanced head and neck cancer) or if they can used to to biologically characterize the disease during its natural history. This review, leaving from the report of the knowledge already consolidated, is devoted to identify the new trends in the research and in the clinical application of CTC, both in locally advanced/localized prostate and breast cancer, and in head and neck cancer.

© 2015 ACT. All rights reserved.

Key words: Circulating tumour cells; Breast cancer; Prostate cancer; Head and neck cancer; Review

Buglione M, Triggiani L, Pasinetti N, Costa L, Pedretti S, Maddalo M, Borghetti P, Almici C, Grisanti S, Magrini SM. Circulating Tumour Cells in Clinical Practice: Can Lessons Learned From Locally Advanced/Metastatic Breast and Prostate Cancers Guide Their use for other Cancer Types? Lights and Shadows. Journal of Tumor 2015; 3(3): 322-342 Available from: URL: http://www.ghrnet.org/index.php/JT/article/view/1506

The cells probably involved in tumour progression

The process of metastatic diffusion of cancer cells is not completely understood. There are a lot of hypotheses and many different, incompletely defined cells are probably involved.

Cancer stem cells (CSC) are characterized by properties conferring them the ability to induce cancer, to survive to the treatment and to produce the regrowth of the tumor both at the primary site and in distant ones[1,2]

Disseminated tumour cells (DTC) have been identified in the bone marrow and may be a marker of minimal residual disease (a concept defined for hematologic neoplasms and not yet directly demonstrated for other solid tumors)[3]

Circulating tumour cells (CTC) probably represent an intermediate step between these two cell types. They can be defined as cells shed from the primary tumour and circulating in the bloodstream. They could be responsible, together with other host factors, of the metastatic process. CTCs are very rarely picked up in the blood; they can be identified using different methods[4, 5].

Only one of these is considered as a standardized and repeatable method. This method identifies cells characterized by EpCAM, Citokeratin 8, 18 and 19, DAPI positivity and CD45 negativity as CTC[6]. The problem of this method is that it could miss cells that are transforming themselves (from epithelial to mesenchymal). The epithelial to mesenchymal transformation (EMT) has been recognised as one of the steps involved in the metastatic process[7-9].

Moreover, the group at MSKCC suggested the role of CTC in the self-seeding process by using an animal model (2009). This hypothesis could explain the occurrence of late local relapses[10,11]. According to this model, a particular type of “aggressive CTC” could be the cause of both local relapse and metastatic disease. This is not really conclusively proven and different authors cast some doubts about this conclusion.

The identification and the recognition of these different types of cells (CSC, DTC and CTC) could give to the physician the possibility to monitor and characterize disease before and during the treatment, as a kind of “liquid biopsy”[12-15].

CTC’s identification has already been demonstrated to have a prognostic and predictive value in metastatic breast, prostate[16,17].

However, in the daily clinical activity we are very far from this. There are still a lot of open questions to be solved before CTC determination can be applied in clinical practice.

Among them:

- can CTCs be used to predict survival also in non metastatic, locally advanced tumours (in particular breast, prostate cancer)?

- can CTCs be used in other tumours (e.g. head and neck cancer)?

- can CTCs be used to biologically characterize the disease during its clinical evolution?

Breast cancer

Breast cancer is the more frequent tumour affecting females. Nowadays, most of these tumours are diagnosed in an early stage, but a lot of them can progress to metastatic disease. Many different therapeutic options (chemotherapy, hormonal and target therapy) are available to treat and to prolong survival of these patients.

The already available prognostic and predictive factors are patient-related (menopausal status, age) and disease related (hormonal status, disease stage, biological factors as Her2 expression). In metastatic patients the absence/presence of visceral disease has a relevant prognostic value. Tumour markers are often used to monitor the evolution of metastatic disease and its response to the treatment; unfortunately, these factors cannot be used to accurately predict progression free and overall survival during treatment[18]. In fact, they cannot anticipate the duration of response to therapy and their changes often do not mirror the clinical evolution of disease.

The discovery of DTC and CTC led clinical investigators to hypothesize they could solve the problem of “anticipating response” to treatment in patients with metastatic breast cancer. Similarly, the identification of DTC or CTC could be used to select patients with localized or locally advanced breast cancer requiring a more aggressive treatment.

Clinical relevance of CTC in metastatic breast cancer

CTC have already been identified as a relevant prognostic factor in metastatic breast cancer. The first evidence of their role was published by Cristofanilli in 2005[19]. In patients submitted to chemotherapy for metastatic breast cancer, he demonstrated the prognostic value of the presence (cut-off value, 5 CTC/7.5 ml) and of the variation in number (increase or reduction) of CTC’s during chemotherapy. In 2006[16] he concluded that the changes of number of CTC’s during the treatment can predict survival and response better than and before than conventional staging by imaging. CTC’s prognostic value in metastatic breast cancer was lastly confirmed in a recent retrospective analysis collecting the data of 1944 patients[20]; 991 of them had ≥ 5 CTC/7.5 ml before chemotherapy and worse progression free and overall survival; similarly, an increase of CTC’s during weeks 3-5 and 6-8 after the beginning of chemotherapy was related to worse progression and overall survival. CTC count during the treatment has a better prognostic value than other tumour markers (CA 15.3 and CEA). Many prospective/retrospective/observational studies confirmed these data. The more recent reports are listed in Table1.

An additional role of CTC identification in metastatic cancer can be the characterization of the differences between primary disease and metastatic disease in terms of Her2 and ER expression; this could lead to understand if the metastatic process changed the “type” of tumour and consequently to select different treatment options according to these findings[15].

The data about the difference in HER2 over-expression between primary tumour and the CTC’s detected in metastatic patients point to the same conclusion (<15% in primary tumour vs 15-90% in CTC’s); the different percentages of CTC’s positivity are probably related to the methods used to identify and characterize the CTC itself[24-28]. Two clinical prospective trials are addressing the possible benefit of using trastuzumab and lapatinib to treat patients with HER2 negative metastatic breast cancer (MBC) but harbouring Her2 positive CTC[29].

The data about ER expression in CTC’s are scarce; CTC’s ER positivity range between 18 to 57 %, a clearly lower value than in the primary tumour (70%)[30-31]; again, differences between studies can be related to the different identification and determination methods. The predictive role of ER-CTC status is, anyway, not clear, since clinical studies are still lacking.

The possible role of CTC’s in locally advanced/localized breast cancer

The clinical significance of CTC’s in locally advanced, localized breast cancer is far from being clear.

Their use has been investigated in different retrospective and prospective trials based on the hypotesis that CTC’s could be an early signal of residual disease and that they could initiate the metastatic process, consequently changing the natural history of the disease.

In fact, it would be very useful if early CTC’s detection could help in defining the prognosis of patients with early, node negative breast cancer patients, identifying those deserving a more aggressive treatment.

Some trials demonstrated that CTC detection is related to a worse disease free, metastases free, cancer specific and overall survival (Table 2).

Adjuvant chemotherapy and CTC

In his German trial (SUCCESS trials) Rack demonstrated in 2010 and more recently[32-33] the relevance of CTC detection (cut-off ≥1) before and after adjuvant chemotherapy, to predict DFS and OS, also as an independent factor.

Neoadjuvant chemotherapy and CTC

Contrasting results are evident regarding the use of CTC’s before and after neo-adjuvant chemotherapy. In patients submitted to neo-adjuvant chemotherapy CTC’s evolution during treatment is independent from the primary tumour response and it is not associated to any of its clinical and pathological features; some studies relate CTC’s reduction with a peculiar pattern of relapse[34].

An unexpected finding is that in some studies CTC’s status after neo-adjuvant chemotherapy does not seem to have any impact on prognosis[35]; in other reports, it relates with a worse DFS if the CTC’s number is ≥1 and with a worse OS if their number is ≥5[34].

A still unsolved problem is how the information from clinical studies can really help the physician to improve survival also in patients with CTC positive determination. The future should again rely on the biological characterization of these cells in order to guide a tailored treatment.

Can CTC be used to guide treatment in non-metastatic breast cancer?

The problem of understanding if CTC’s can be used to change the treatment of patients with localized breast cancer is not a little one. The results from the only study already published seem to show that an early change of treatment in patients with an increasing CTC number does not translate in a better OS: they maintain a prognosis worse than that of patients without CTC or with a reduced number of CTC after 21 days of treatment[21].

Different prospective clinical trials are now on-going to give an answer to this strategic question.

Table 3 shows only the trials registered at the clinicaltrials.gov website.

Prostate cancer

Prostate cancer is most common tumor in males. It is often discovered occasionally and as a very localized disease (low-risk disease). For these patients the therapeutic options can vary from “watchful waiting” to radiotherapy alone to surgery alone depending also by patient preference.

The patients defined at high risk (locally advanced tumour, high Gleason score and high PSA level)[40] are usually treated with conservative treatment: radiotherapy (with or without pelvic lymph-nodal irradiation[41] depending on the risk of nodal involvement and clinical practice of the different institutions) and hormonal treatment, better if more than two years[42].

The usual medical treatment of high risk or early metastatic disease is hormonal therapy (LHRH analogue or total androgenic block).

A substantial proportion of high risk patients progresses to metastatic disease and can become resistant to hormonal treatment.

There are no clear methods, apart from the already mentioned prognostic factors, to predict the clinical evolution of disease and to identify patients to be consequently treated with the more aggressive treatment options.

The rationale suggesting clinical investigators to search disseminated or, better, circulating tumour cells in the bone marrow or in the blood, respectively, also in prostate cancer is the same already discussed for breast cancer: to identify a simple, more effective method to predict survival and to guide the treatment.

The evidence about CTC prognostic value, however, is weaker for prostate than for breast cancer.

CTC in metastatic hormone sensible (HSPC) or castrate resistant (CRPC) prostate cancer

It has been demonstrated that CTC are detectable in patients with metastatic PC; the number and the evolution of CTC number during treatment is significantly related to survival and response to therapy.

The results of the recently published SWOG-S0421 trial confirmed the prognostic importance, in terms of OS, of CTC enumeration and change in number between baseline and day 21 in a phase III trial of patients treated with docetaxel with or without atrasentan[43]. Median CTC number at day 0 was 5 cell; a CTC number lower and higher than 5 was associated with PSA levels, bone pain, liver disease, haemoglobin level, alkaline phosphatase and PSA and RECIST response. OS was equal to 26 months for patients with < 5 CTC and 13 months in those with >5 CTC; a raising count between days 0 and 21 was associated with shorter OS. Less recent phase II studies showed the same results in terms of number of CTC before the treatment[44-45] and change of the number during the treatment[46]. These results induced FDA to authorize the use of CTC detection for the evaluation of metastatic CRPC using a standardized method.

The subsequent step will be to verify the utility of CTC count as surrogate of survival in CRPC. The requisite to consider a parameter as a response-indicator biomarker are very restrictive and has to be confirmed and re-confirmed in prospective randomized trials[47].

In HSPC only a few little studies suggest the utility of CTC. In 2011 Goodmann[48] demonstrated that CTC number is directly related to ALP and LDH levels and not related to PSA and testosterone levels (33 patients); at multivariate analysis only CTC at baseline had independent predictive value in terms of duration and magnitude of response to hormonal therapy. In 2012 Folkersma[49] compared 30 pts with localized PC, 30 patients with metastatic PC and 30 healthy volunteers. A significant correlation between all the marker of disease extension but Gleason score was demonstrated (PSA, T, N, M class). A prognostic correlation in terms of OS and PFS was evident if CTC count was >4; these data were confirmed at multivariate analysis.

It is still not clear if CTC enumeration can be used as an early marker of the evolution of disease and to early predict the loss of hormonal sensibility. More data are necessary to confirm this data and to allow a practice of change of treatment based on CTC count.

Localized prostate cancer

CTC appear to be less frequently detectable in localized than in metastatic PC. The studies in this patient setting are scarce; recently[50] it has been demonstrated that only 5/36 patients with newly diagnosed high risk PC (age >70, median PSA 14.1, median GS 9) had CTC in their blood. The presence of CTC did not correlate with other already confirmed prognostic factors (PSA, GS, stage T). Same results were evident comparing the percentage of CTC positivity between control subjects (20) and patients (20) with locally advanced PC (median PSA 21ng/ml, T>cT3a) with a p=0.66[51].

The low frequency of CTC positivity in localized prostate cancer forced clinical investigators to search different methods to identify CTC: CTC-chip platform[52], RT-PCR for prostate specific genes as a surrogate for counting CTC and as a mean to predict PC recurrence after prostatectomy[53].

Lowes in 2012[54] investigated the role of CTC in patients treated with radical radiotherapy (RT) for locally advanced PC and did not found a reduction of CTC number after RT; however, PSA responders could have a more pronounced reduction of CTC number than non responders (even if this observation does not reach statistical significance).

Again in this setting of patients the correlation between CTC and DTC in the bone marrow has been looked for, but the correlation was difficult to demonstrate. DTC itself were associated with worse prognosis in terms of probability of metastatic disease (multivariate analysis)[55-58].

Nowadays it is still not clear if the simple enumeration of CTC with standardized methods could be used in clinical practice for localized prostate cancer, either as a prognostic or a predictive factor.

Molecular characterization of CTC

The future studies on CTC in prostate cancer will probably address the biological characterization of these cells, in order to directly target the treatment to specific and different types of prostate cancer. A lot of different methods can be used to characterize the genomic and proteomic pattern of CTC including immunohistochemistry, immunofluorescence (presence or loss of phosphatase and tensin homolog PTEN; assess the number of copy of the AR gene), gene copy number analysis (detection of DNA mutations and altered copy number), genome sequencing analysis (hotspot mutation of phosphoinositide 3-kinase PI3K/AKT genes in the circulating genomic material) and epigenetic studies[59].

The interest of these different methods to track biological targets is the opportunity to monitor the disease during its natural history, to identify the evolution of the tumor in response to different treatment, to try to develop specific targeted treatments.

Clinical trials addressing future developments in CTC use

The lack of definitive answers as far as the clinical impact of CTC measurement is concerned is also testified by the ongoing trials reported in Table 4.

The main goals of these trials are: the choice of the better method to identify CTC; the clinical advantages of the use of newer identification methods; the impact of molecular characterization of CTC on survival.

How to apply the lessons learned from breast and prostate cancers to other cancer types: the case of head and neck tumours (HNC)

The interest for a possible relationship between CTC count and outcome in head and neck cancer has been recently growing because of the difficulties in predicting survival with the already known prognostic factors.

Different combinations of surgery, radiotherapy and chemotherapy are the mainstay of the treatment of head and neck cancer. The choice of the actual treatment derives from the tumor site (naso-, meso-, hypo-pharynx, larynx) and, within each site, from T, N and M categories and from their interplay, defining disease stage. Surgery is often the first approach, but radiotherapy is very often chosen to also in locally advanced cases, aiming at the cure of the disease with less functional damage[60].

Overall and progression free survival times after primary treatment can be predicted using mainly the same parameters (T, N, M).

Only recently some biological factors have been added to the diagnostic armamentarium as prognostic and predictive factors. The most frequently used biological predictors are the EGFR status (since EGFR is the target for some effective drugs, like cetuximab, and even if EGFR positivity is overwhelmingly frequent in HNC)[61, 62]; and the HPV positivity, mainly for meso-pharyngeal cancer, identifying patients with a better outcome even after less aggressive treatments[63]. The evidence on the predictive efficacy of these markers is growing, but they cannot be considered a standard mean to drive treatment choice.

Locally advanced disease

The interest about CTC determination in head and neck cancer is grown only in the last two-three years.

The research steps were not dissimilar from the case of breast and prostate cancer: 1. Try to identify CTC in the blood; 2. Try to correlate their presence and number to the other already known prognostic and predictive factor; and finally 3. Try to understand if they could more effectively predict response to treatment and prognosis.

Differently from the case of breast and prostate cancers, early studies were focused on locally advanced disease.

The results were not uniform.

Hristozova did not found a statistically significant correlation between CTC identification, T stage and tumour volume; an association with N2b status was however reported[64]; Bozec did not find any correlation with the already known prognostic factors (T, N, M)[65]; Buglione found an higher frequency of CTC detection in patients with stage IV disease, as compared with earlier stages, but without statistically significant differences[66].

The percentage of CTC positivity before primary treatment ranges between 15% and 43% and it is strongly dependent from the search method, being lower with standardized methods (Table 5)

Some authors demonstrated differences between CTC identification in different sites and also different clinical implications for CTC detection according to the tumor site: for example, a favourable prognostic significance in oro-pharyngeal cancer and a negative prognostic impact for the other disease sites have been reported[67] Our group detected CTC more frequently in oro- and hypo-pharyngeal cancer and SNUC cases[66].

Recently Gröbe[68] in a selected series of patients treated with surgery for oral cancer demonstrated a correlation between T, N, M category, CTC detection rate (in blood samples) and DTC positivity (in bone marrow). CTC and DTC were present in 12.5% and 20% of the patients, respectively. It is very interesting to note that CTC and DTC were identified together only in two patients. He demonstrated, both at univariate and multivariate analysis a statistically significant correlation between CTC/DTC detection and recurrence free survival.

As far as survival is concerned, again, the results reported are not uniform. Buglione[66] reported significantly better complete/partial response rates in patients without CTC or with a decreasing number of CTC during the treatment; no statistical evaluation of DFS or OS was done because the follow up period was too short. A decrease in the CTC number or their absence throughout the treatment was related with non-progressive disease, after both complete or incomplete remission, and with the number of patients alive and NED (no evidence of disease) (p = 0.009). Tinhofer[67] demonstrated that CTC presence was not predictive of OS or DFS in the entire series (144 patients); in oro-pharyngeal carcinomas (OPC, 63 patients) CTC detection was associated with improved DFS (CTC+ vs CTC-: 100% vs. 79%; p=0.059); the reverse was true for carcinomas of other sites (n=81; CTC+ vs CTC-: 29% vs 75%; p=0.001). In multivariate analysis, CTC remained an independent prognostic marker for DFS (p=0.002) and OS (p=0.016) in non-OPC.

Only one study tried to relate CTC presence to its biological characterization and to treatment response[69]. The number of CTC-positive samples significantly increased after radiotherapy. This radiation-induced increase in CTC numbers was less pronounced when radiotherapy was combined with cetuximab compared to the combination with cisplatin/5-fluorouracil. The Cetuximab regimen was also effective in reducing phosphorylate EGFR (pEGFR) expression in CTCs.

Metastatic disease

More than 50% of the cases with stage III-IV head and neck cancer recur with metastatic disease. One third of all treated patients will respond to systemic treatment with complete or – more often- partial remission, even if cure is almost never obtained. It is difficult to predict both which patients will recur with metastatic disease and which patients will respond to the treatment. Only one (not validated) prognostic score has been published to try to predict progression free and overall survival in metastatic disease. This score considers factors like performance status, weight loss, primary site, response to radiotherapy and tumour cell differentiation[70].

As for breast and prostate cancers, it is possible to use CTC to predict the outcome of patients treated with chemotherapy.

Our group explored also the use of CTC in metastatic patients. Grisanti[71] confirmed the presence of CTC before chemotherapy (26%) and during treatment. He demonstrated that at univariate analysis ≥2 CTCs are related with a prognosis poorer than 0-1 CTC; the presence of ≥1 CTC was related to worse PFS and OS. At multivariate analysis, disease control after systemic therapy was obtained in 8% of CTC-positive patients as opposed to 45% of the CTC-negative ones (p 0.03).

An important research effort will be needed do better define the possible clinical use of CTC in this clinical setting; the data collected, even if interesting, are in fact far from being sound enough to find a clear clinical application.

Also for HNC, the final goal resides in the biological characterization of these cells to try to guide the treatment directly according to specific targets.

Probably because the rarity of this kind of tumours the on-going prospective trials devoted to understand the role of CTC are unfortunately very rare (Table 6).


A lot of scientific and economic energies have been devoted to understand the role of CTC to enhance the knowledge of the natural history of some cancer types.

Metastatic breast cancer was the first disease for which FDA in USA approved the use of CTCs as a prognostic/predictive factor. Metastatic prostate cancer was the second. Despite this important acknowledgement, until now, the use of this new biologic instrument did not impact significantly on the choice of the treatment.

CTC determination in metastatic breast and prostate cancer can be effective when used as prognostic and predictive factor. We know that the presence of CTC before the beginning of the treatment correlates with worse progression free and overall survival, but neither the knowledge of its change during the treatment neither the early change of treatment guided by CTC determination seem to modify the outcome.

Results are even scarcer in locally advanced and early breast and prostate cancer. For early disease, questions far outweigh answers. Correlations between CTC determination and the other already used prognostic factors remain unclear and it is impossible to use CTC determination to guide treatment choice.

The reason of these difficulties probably depends from the methods of determination (probably not all the cells are correctly identified) and from the lack of biologic characterization. In locally advanced and early stage disease ineffective detection methods probably are not able to identify the few, possibly present, CTC.

In addition, disease biological features change during its natural history; the treatment should be probably targeted on these new targets rather than on those of disease at presentation.

The ongoing trials in breast and prostate cancer are trying to answer these open questions in order to confirm or discard, also from an health economics point of view, the utility of CTC determination and characterization.

The problem is obviously much more complex for head and neck cancer.

Difficulties with HNC are amplified by the heterogeneity of the disease(s) in terms of staging and prognosis (epi-pharyngeal tumor are very different from meso-pharyngeal or hypo-pharyngeal or laryngeal tumors and all they are different from oral cavity tumors) and treatment (different combinations of surgery, radiotherapy and chemotherapy or biological treatment).

Results on correlations between CTC determination and stage, site of primary are therefore very different in the different trials. The percentage of CTC positivity varies very much between the different trials and according to the the different methods of determination.

The results of the only prospective trial on metastatic disease confirm the prognostic role of CTC before the beginning of chemotherapy and the significance of CTC reduction during the treatment, in terms of disease control.

CTC determination has been applied also to other neoplasms, not covered by this review. Among them at least lung cancer should be quoted[74-76].

However, in an era of reduced resources for health systems it is easily understandable that very costly predictive and prognostic tests should be fully validated before being adopted in the routine clinical practice.

The study of CTC has important biological implications; it could advance further our knowledge of the neoplastic phenotype and of its variations after therapy. However, clinical applications are in their infancy and should be considered very cautiously. This does not mean that research in this field should be abandoned; rather, more sound biological bases should probably be built before attempting extensive clinical testing.


Prof Piero Nicolai, Prof Roberto Maroldi, Prof Alfredo Berruti, Dott.ssa Mirella Marini, Dott.ssa Monica Mangoni


There are no conflicts of interest with regard to the present study.


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