Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer
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Circulating Tumor Cells, Disease
Progression, and Survival in Metastatic Breast Cancer
Massimo Cristofanilli
Metastatic breast cancer (MBC) is considered incurable; therefore, palliative treatment is
the only option. The biologic heterogeneity of the disease is reflected in its somewhat
unpredictable clinical behavior. The presence of circulating tumor cells (CTCs) in patients
with MBC about to start a new line of treatment has been shown to predict progression-free
and overall survival. This prognostic value is independent of the line of therapy (eg, first or
second line). Moreover, a multivariate analysis has shown the prognostic value of CTCs to
be superior to that of site of metastasis, type of therapy, and length of time to recurrence
after definitive primary surgery. These data suggest that the presence of CTCs may be used
to modify the staging system for advanced disease. Larger studies are needed to confirm these
data and evaluate the use of CTC detection in monitoring treatment and furthering our
understanding of breast cancer biology when combined with other diagnostic technologies.
Semin Oncol 33(suppl 9):S9-S14 © 2006 Elsevier Inc. All rights reserved.
B reast cancer is one of the most frequently diagnosed types
of cancer and a leading cause of cancer death in women.1
The vast majority of these deaths are caused by recurrent
tastasis is a highly selective competition favoring the survival
of a minor subpopulation of metastatic tumor cells. These
findings suggest that the use of novel, sophisticated diagnos-
metastatic disease. Occult dissemination of tumor cells is the tic technologies can allow early identification of micrometa-
main cause of recurrent metastatic breast cancer (MBC) in static foci, providing an opportunity for early intervention in
patients who have undergone resection of their primary tu- patients at high risk, and a better risk stratification in patients
mor.2 Approximately 5% of patients with breast cancer have with metastatic disease, leading to appropriate treatment de-
clinically detectable metastases at the time of initial diagnosis. velopment and patient selection.
A further 30% to 40% of patients who appear clinically free of The detection of microscopic disease in breast cancer has
metastases harbor occult metastases.3,4 been evaluated in lymph nodes, bone marrow (primary
The formation of metastatic colonies is a continuous pro- breast cancer), and peripheral blood (metastatic disease).3-6
cess, commencing early during the growth of the primary Most of these studies have shown that the detection of mi-
tumor. Metastasis occurs through a cascade of linked sequen- croscopic disease in patients with breast cancer contributes
tial steps involving multiple host-tumor interactions. To suc- prognostic information and, in selected cases, can predict the
cessfully create a metastatic deposit, a cell or group of cells efficacy of treatments.6 In primary breast cancer, the detec-
must be able to leave the primary tumor, invade the local host tion of microscopic disease in lymph nodes and bone marrow
tissue, and survive to proliferate. This complex process re- has led to a better understanding of the role of minimal re-
quires the cells to enter the circulation, arrest at the distant sidual disease. The detection of tumor cells in the locore-
vascular bed, extravasate into the organ interstitium and pa- gional lymph nodes of women with early breast cancer using
renchyma, and proliferate as a secondary colony. Several ex- immunohistochemical analysis is technically feasible, and the
perimental studies suggest that during each stage of the pro- presence of these cells has been shown to have a negative
cess only the most fit tumor cells survive.2 A very small effect on long-term prognosis.7 In this review, we specifically
percentage (less than .01%) of circulating tumor cells (CTCs) discuss the detection of microscopic disease in the peripheral
ultimately initiate successful metastatic colonies. Thus, me- blood, and the prognostic implication and clinical use of
determining the presence of CTCs.
Department of Breast Medical Oncology, The University of Texas M. D.
Anderson Cancer Center, Houston, TX. Detection of CTCs in MBC
Address reprint requests to Massimo Cristofanilli, MD, FACP, Department
of Breast Medical Oncology, Unit 1354, The University of Texas M. D. The first report on tumor cells in the peripheral circulation
Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030- was attributed to Ashworth in 1869.8 Since then, the exis-
4009. E-mail: mcristof@mdanderson.org tence, origin, and clinical significance of CTCs have been
0093-7754/06/$-see front matter © 2006 Elsevier Inc. All rights reserved. S9
doi:10.1053/j.seminoncol.2006.03.016S10 M. Cristofanilli
debated. The introduction of more sensitive and specific im- value of CTCs in patients with MBC who were about to start
munohistochemical techniques in the late 1970s led to re- a new systemic treatment. The 177 enrolled patients under-
newed interest in the detection of CTCs and their possible went blood collection at monthly intervals for up to 6 months
association with minimal residual disease in solid malignan- after enrollment (Table 1). A cutoff of 5 CTCs/7.5 mL was
cies.9,10 But, despite evidence of the prognostic value of CTCs used to stratify patients into positive and negative groups
in some studies, the detection of micrometastases was never (positive, ⱖ5 CTCs/7.5 mL; negative, ⬍5 CTCs/7.5 mL).
incorporated into cancer staging protocols or considered a Patients classified as positive had shorter progression-free
valuable tool for clinical use. This may be the result of a survival times (2.7 v 7.0 months; P ⫽ .0001) and shorter
combination of factors, such as variable antigen expression in overall survival times (10.9 v 21.9 months; P ⬍.0001) than
poorly differentiated tumors and reports of cytokeratin and did those classified as negative (Fig 1). At first follow-up after
epithelial membrane antigen positivity in nonepithelial cells, initiation of therapy (3 weeks) there was an increased differ-
which showed a need for more sensitive and specific methods ence between CTC-positive and -negative patients in terms of
of detection than were available at the time. This need was progression-free survival time (2.1 v 7.0 months; P ⬍.0001)
fulfilled to some degree by more sensitive polymerase chain and overall survival time (8.2 v ⬎18 months; P ⬍.0001). On
reaction (PCR) techniques in the late 1980s, which greatly multivariate Cox hazards regression analysis, CTC levels,
facilitated the detection of occult tumor cells through the use both at baseline and at first follow-up, were the most signif-
of nucleic acid analysis.11-13 In the past decade, a few studies icant predictors of progression-free survival and overall sur-
vival.6
have shown that the detection of occult disease by PCR has
These data suggest several important considerations. First,
prognostic significance in some solid tumors.14 However, be-
and more relevant to clinical practice, is the demonstration
cause these PCR-based assays for the detection of occult tu-
mor cells have limitations, particularly contamination of
samples, sensitivity and specificity of the assay, and inability
Table 1 Demographics of Patients With Metastatic Breast
to quantify tumor cells, they cannot be used to perform func-
Cancer Before Undergoing New Systemic Treatment
tional assays. These factors have precluded the widespread
use of PCR in in vitro diagnostic applications. N %
Over the past few years, immunomagnetic separation tech- All patients 177
nology, with its higher level of sensitivity and specificity, has Age at baseline (yrs) 58.5 ⴞ 13.4
been used to improve the detection of CTCs.15-17 In this tech- Median (yrs) 59
nique, the specimen is incubated with magnetic beads coated Race
White 152 86
with antibodies directed against epithelial cells. The epithelial
Black 14 8
cells are then isolated using a powerful magnet. The magnetic Hispanic 7 4
fraction can be used for downstream reverse-transcriptase PCR, Unknown 4 2
flow cytometry, or immunocytochemical analysis.18,19 Using ER/PR
this approach, Austrup et al reported the prognostic signifi- ER/PRⴙ 120 68
cance of genomic alterations (eg, c-erbB2 overexpression) ER/PRⴚ 55 31
present in circulating cells purified from the blood of patients Unknown 2 1
with breast cancer.20 The authors investigated genomic im- HER2 status
balances such as mutation, amplification, and loss of het- HER2ⴚ (0ⴚ2ⴙ) 122 69
HER2ⴙ (3ⴙ) 26 15
erozygosity of 13 tumor suppressor genes and two proto-
Unknown 29 16
oncogenes using DNA from isolated minimal residual cancer Line of chemotherapy for
cells. The presence and number of genomic imbalances mea- metastases
sured in disseminated tumor cells were significantly associated 1st 83 47
with worse prognosis.20 More recently, advances in technology 2nd 25 14
have facilitated the detection of extremely rare CTCs. The >3rd 67 38
CellSearch system (Veridex, LLC, Warren, NJ) was designed Unknown 2 1
to detect circulating epithelial cells in whole blood. This sys- Site of metastasis
Visceral 145 82
tem is based on the principle of immunomagnetic isolation of
Non-visceral 32 18
epithelial cells, with subsequent counting provided by im- Survival status
munofluorescent analysis of cytokeratin expression.21,22 Alive 74 42
Dead 103 58
Average [mean] follow-up 19.5 ⴞ 5.7 (median,
Prognostic Role of CTCs Time alive (mos) 20.9 months)
The predictive and prognostic roles of CTCs were recently (N ⴝ 74)
investigated in a prospective multicenter clinical trial led by Average [mean] dead ⴞ SD 10.3 ⴞ 6.9 (median,
researchers at The University of Texas M. D. Anderson Can- (mos) (N ⴝ 103) 8.8 months)
cer Center (Houston, TX).6 In this study, CellSearch was used Abbreviations: ER, estrogen receptor; HER, human epidermal growth
to prospectively determine the prognostic and predictive factor receptor; PR, progesterone receptor; SD, standard deviation.CTCs, disease progression, and survival S11
that detection of CTCs is superior to known factors, such as
site of metastasis (visceral versus non-visceral) and estrogen-
receptor status, that have been previously considered impor-
tant variables for prognostic evaluation. Furthermore, these
results showed that CTCs are detectable in MBC irrespective
of the site of metastasis, the line of therapy (eg, patients who
are newly diagnosed versus those undergoing second- or
third-line therapy), and, more importantly, initial hormone
receptor status. Of note was the fact that the proportion of
patients with newly diagnosed metastatic disease about to
undergo first-line treatment who were CTC-positive (ⱖ5
CTCs/7.5 mL) was similar to the proportion of patients un-
dergoing at least second-line treatment who were CTC-pos-
itive (52% v 48%, respectively).6 The detection of CTCs pre-
dicted overall survival independently of the number of
previous treatments, but the association was particularly
strong for patients undergoing first-line treatment for meta-
static disease (Fig 2).23 The data at first follow-up supported
the predictive value of CTCs. As expected, the CTC detection Figure 2 Kaplan-Meier plots of overall survival in first-line therapy
rate in the group tested at first follow-up was lower than the MBC patients with ⬍5 CTCs or ⱖ5 CTCs (per 7.5 mL) at baseline.
baseline, particularly in patients undergoing first-line treat- The analysis includes 94 patients with measurable and evaluable
ment (25% v 52%) and those with visceral disease (28% v (bone only) disease. Overall survival was calculated from the time of
the baseline blood draw. Coordinates of dashed lines indicate me-
dian survival time. CTCs, circulating tumor cells; CI, confidence
interval; HR, hazard ratio; OS, overall survival.
50%).6 These data indicate that patients with newly diag-
nosed disease who are about to start first-line therapy may
have detectable CTCs, and the changes in these detection
rates at 3 to 4 weeks may indicate benefit from the use of
systemic treatment (particularly chemotherapy). However,
this short follow-up testing might be less useful in patients
who presumably have more indolent disease (eg, patients
undergoing hormonal treatment). A recent update and re-
analysis of the data from this pivotal study, which included
data from an additional 46 patients with bone-only MBC,
confirmed the observation in the initial report indicating that
the detection of CTCs at baseline was associated with a sig-
nificant cumulative hazard risk of death (53% v 19% with
and without CTCs, respectively; P ⫽ .0001) in patients with
measurable as well as ‘nonmeasurable’ MBC.24
In summary, the detection of CTCs in patients with MBC is
associated with important prognostic implications, allowing
for a defined stratification of risk of death. These findings
suggest that CTC detection can be used for a stage subclassi-
fication of MBC that would modify our approach to treatment
planning and, more importantly, allow for more sophisti-
cated design of efficacy trials.
Staging Classification
for MBC Using CTCs
Figure 1 Kaplan-Meier plots of (A) progression-free survival and
(B) overall survival in MBC patients with ⬍5 CTCs or ⱖ5 CTCs (per The American Joint Committee on Cancer (AJCC) classifica-
7.5 mL) at baseline. Progression-free survival and overall survival tion system includes much of the traditional prognostic in-
were calculated from the time of the baseline blood draw. Coordi- formation used by clinicians when developing a comprehen-
nates of dashed lines indicate median survival time. CTCs, circulat- sive treatment plan. This system was based on a schema
ing tumor cells. developed by the Union Internationale Contre le CancerS12 M. Cristofanilli
(UICC).25-27 In brief, the AJCC system attempts to define the
disease by incorporating all aspects of cancer distribution in
terms of the primary tumor (T), lymph nodes (N), and distant
metastasis (M). A ‘stage’ group (0, I, II, III, or IV) is then
assigned on the basis of the possible TNM permutations, with
0 reflecting minimal involvement and IV either the most tu-
mor involvement or distant metastasis. This basic TNM stag-
ing is then further subdivided according to the time the eval-
uation is performed: c ⫽ clinical (before surgical treatment)
and p ⫽ pathologic (after surgical specimen analysis).7,28 Figure 3 Schematic representation of the prospective International
Over the past 45 years, the AJCC has regularly updated its Stage IV Stratification Study. Six hundred sixty patients with newly
staging standards to incorporate advances in prognostic tech- diagnosed MBC will be enrolled and, after having blood drawn at
nology. The committee’s current work concerns the develop- baseline, will be receiving treatments. Patients will have follow-up
ment of prognostic indices based on molecular markers. But of their condition until death. There is no planned interim analysis
until these changes are incorporated, TNM staging quantifies or additional blood evaluation. Dx, diagnosed; MBC, metastatic
only the physical extent of disease and, although it includes breast cancer; Tx, treatment.
the approximately 2% of women who initially present with
primary metastatic disease (stage IV), the prognostic infor-
mation is only applicable to in situ, local, and regional pri-
Thus, the main goal in the management of this entity is pal-
mary breast cancers. This is somewhat problematic in that
liation.29,30 Only a few patients who experience a complete
because of the heterogeneity of the disease, the potential for
response after chemotherapy remain in this state for pro-
continued mutation, and the variety of treatment options
longed periods, with some remaining in remission for more
available, the information acquired at the time of the primary
than 20 years.30 These long-term survivors are usually young,
diagnosis of breast cancer may not be as relevant to planning
have an excellent performance status, and, more importantly,
the treatment of the approximately 30% of women with
have limited metastatic disease.31,32 Most patients with met-
breast cancer who present with recurrent MBC years after
astatic disease respond transiently to conventional therapies
their initial diagnosis.
and develop evidence of progressive disease within 12 to 24
The recent demonstration that the presence of CTCs pre-
months of starting treatment.29 For these patients, systemic
dicted the prognoses of two subgroups of patients with MBC
raises the possibility that this method will allow for a true treatment does not result in a significant improvement in
‘biologic staging’ of breast cancer (eg, stages IVA and IVB).6 survival time but substantially improves their quality of life.
The main limitations of the previous completed study were At the present time, clinicians can use three different
the sample size (only 177 patients), the inclusion of measur- systemic treatment modalities for advanced breast cancer:
able-disease-only patients, which did not reflect the real clin- endocrine therapy, chemotherapy, and biologic targeted
ical heterogeneity of MBC, the inclusion of patients at differ- therapy.33-35 Appropriate selection of patients for these mo-
ent points in treatment, and those who have newly diagnosed dalities is based mostly on tissue assessment of hormone
disease versus those undergoing second- or third-line treat- receptor status (estrogen and progesterone receptors) and
ment, whose benefit from further therapy could have been c-erbB2 status. In patients who lack expression of hormone
limited by the advanced status of their disease. To overcome receptors or who show no amplification of c-erbB2, cytotoxic
these limitations, a larger international validation trial, the chemotherapy is used. However, no standard therapeutic
International Stage IV Stratification Study, has recently be- regimen has been defined. For example, the optimal schedule
gun (Fig 3). The objective of this study is to confirm the of chemotherapy administration in MBC (ie, concurrent v
prognostic value of CTC detection in a larger and more ho- sequential) remains controversial, and the decision must be
mogeneous cohort of patients with newly diagnosed MBC. individualized. Sledge et al36 addressed this issue in a pro-
The study aims to enroll 660 patients and will enable a more spective study that included 739 chemotherapy-naive pa-
detailed analysis of the association between CTC detection tients who were randomly assigned to receive, at progression,
and other factors, such as ethnicity (black compared with doxorubicin, paclitaxel, or a combination of both. Although
white and Hispanic groups) and specific disease sites (vis- the response rates and times to treatment failure were im-
ceral v non-visceral disease). Accrual is expected to be com- proved with the combination regimen, the overall survival
pleted by November 2006, which will allow completion of rate was comparable between the groups. Other trials have
data analysis by late 2007. This study may well help provide shown a survival advantage for combination regimens, but all
definitive information on the subclassification of stage IV disease of these studies have reported differences in median overall
for the revised 7th edition of the AJCC’s Cancer Staging Manual. survival time, suggesting that even with comparable inclu-
sion criteria the heterogeneity typical of MBC cannot be elim-
inated.37,38 This is indeed one of the major limitations to the
Clinical Application of CTCs development of more personalized treatments for patients.
Despite years of clinical research, the odds of patients with Therefore, although the data show that patients can benefit
MBC achieving a complete response remain extremely low. from combination therapy, they do not clearly identify theCTCs, disease progression, and survival S13
subsets of patients who most benefit or who experience only tional, with decreased levels of major histocompatibility
additional toxicity. complex class II, CD86 (B7) expression, and interleukin-12
In this context, the use of CTCs to stratify patients at the secretion.49
time of disease recurrence may be appropriate. CTC detec- The inherent and dynamic phenotypic instability of breast
tion may allow a more rational selection of treatments for tumor cells complicates these issues of breast cancer biology.
patients with newly recurred disease, and this approach Because tumors arise endogenously and progress, a continual
could maximize the chance of a particular combination or dialogue between the tumor phenotype and the immune re-
single new drug showing clinical benefit and, eventually, sponse ensues, with each influencing the other to evolve.50
prolonging survival. In essence, it is possible that CTC detec- Therefore, being able to evaluate both the phenotype of CTCs
tion could be used in the design of efficacy trials of different and the related immune response is a critical step in under-
therapeutic approaches. The efficacy of these treatments will standing the complexity of tumor immunity in patients with
be more easily assessed when patients have been stratified by breast cancer, and will enable more effective vaccine strate-
their prognosis, leading to more tailored treatment strategies. gies to be designed. A recently developed glycan array has
We believe that the challenge for the next generation of clin- shown the capacity to detect a particular profile of antibodies
ical trials, and the responsibility for both clinical investigator directed against cancer-specific carbohydrate antigens.51,52
and the pharmaceutical industry, will be to incorporate these The further development of this novel technology in combi-
concepts into the process of drug development. nation with CTC detection will represent a new frontier in
breast cancer microdiagnostics.
Future Uses of CTCs
The process of sorting cancer cells from other cellular com- Conclusion
ponents (eg, blood and stromal cells) in clinical samples is The detection of microscopic disease in the peripheral blood
fundamentally important for the future of genomic and pro- of patients with MBC is associated with prognostic informa-
teomic analysis.39-42 Indeed, the feasibility of evaluating the tion. These data will allow appropriate risk stratification and
gene expression profiles of cancer cells depends mostly on modification of the current staging system for advanced dis-
the quality of the specimen (relative proportion of cancer ease. Moreover, it is expected that investigators and the phar-
cells) and the amount of nucleic acid that can be extracted maceutical industry will derive benefit from these technolo-
from the appropriate cells to provide mRNA suitable for anal- gies that will contribute to more tailored treatments and
ysis.41,42 Fine-needle aspiration biopsy of malignant lesions is sophisticated trial designs. Using a combination of CTC de-
frequently used for diagnostic purposes, but the percentage tection and other diagnostic technologies will help us further
of malignant cells recovered with this procedure ranges understand the complex and heterogeneous tumor pheno-
widely (60% to 90%).41,42 Pusztai et al42 showed that single- type and its relationship with tumor-related immunity.
pass fine-needle aspiration biopsies can provide samples con-
sisting of approximately 79% cancer cells (range, 25% to
100%).
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