Circulating Tumor Cells: Not All Detected Cells Are Bad and Not All Bad Cells Are Detected

Abstract

More than 90% of cancer deaths result from the development of hematogenously disseminated metastasis. The presence of circulating tumor cells (CTCs) in patients with cancer was first reported in 1869. 1 More recently, methods have been developed to detect, isolate, and characterize CTCs in multiple different malignancies that arise in solid organs. 2 The most widely used method, designated CellSearch (Veridex, Raritan, NJ), relies on immunomagnetic capture of CTCs using antibodies against the epithelial cell adhesion molecule (EpCam), which is expressed on the cell surface of many epithelial malignancies, followed by additional characterization with 4,6-diamidino-2-phenylindole staining (to

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DOI: 10.1200/JCO.2010.34.0745; published online ahead of print at
www.jco.org on March 21, 2011
■■■
Circulating Tumor Cells: Not All Detected Cells Are
Bad and Not All Bad Cells Are Detected
Max S. Wicha and Daniel F. Hayes, The University of Michigan Comprehensive Cancer Center, Ann Arbor, MI
See accompanying articles on pages 1547 and 1556
More than 90% of cancer deaths result from the development
of hematogenously disseminated metastasis. The presence of cir-
culating tumor cells (CTCs) in patients with cancer was first re-
ported in 1869.
1
More recently, methods have been developed to
detect, isolate, and characterize CTCs in multiple different malig-
nancies that arise in solid organs.
2
The most widely used method,
designated CellSearch (Veridex, Raritan, NJ), relies on immuno-
magnetic capture of CTCs using antibodies against the epithelial
cell adhesion molecule (EpCam), which is expressed on the cell
surface of many epithelial malignancies, followed by additional
characterization with 4⬘,6-diamidino-2-phenylindole staining (to
demonstrate that the detected event is a nucleated cell), and by immu-
nofluorescence analysis with antibodies against cytokeratin (to dem-
onstrate it is epithelial) and CD45 (to demonstrate it is not a
leukocyte).
2
Numerous studies have shown that the presence of ele-
vated CTC levels, as determined by CellSearch, is negatively correlated
with prognosis in patients with metastatic cancers of the breast,
3,4
prostate,
5
and colon.
6
These studies have demonstrated the analytic and clinical
validity, as defined by the Evaluation of Genomic Applications in
Practice and Prevention Initiative of the Centers for Disease Con-
trol,
7
of counting CTCs in these common solid malignancies.
However, the clinical utility of monitoring CTC levels remains
controversial. Although the US Food and Drug Administration has
cleared the CellSearch assay for clinical use, the American Society
of Clinical Oncology Tumor Marker Guidelines Committee has
not recommended incorporation of CTC levels by any method into
standard care of patients with metastatic breast cancer.
8
Nonethe-
less, studies published subsequent to that analysis seem to support
a limited role for evaluation of CTC levels. In patients with meta-
static breast, colon, and prostate cancers, these assays might be
used to determine if a patient who has been receiving a given
regimen for some time has progressive disease and would be better
treated with an alternative regimen. Furthermore, the Southwest
Oncology Group is conducting a prospective randomized clinical
trial (S0500; A Randomized Phase III Trial to Test the Strategy of
Changing Therapy Versus Maintaining Therapy for Metastatic
Breast Cancer Patients Who Have Elevated Circulating Tumor Cell
Levels at First Follow-Up Assessment) that is designed to test the
clinical utility of changing therapy for patients with metastatic
breast cancer who have not cleared CTCs after only one cycle of a
new, first-line chemotherapy.
2
The use of any assay for CTCs in early-stage solid malignan-
cies has been limited by the poor performance characteristics in
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this setting: low sensitivity and poor specificity. Only approxi-
mately 10% of patients with stage I and II breast cancer have ⱖ1
cell/23 mL whole blood, a lower cutoff in a larger volume than was
chosen to be optimal in the metastatic setting (ⱖ5 CTCs/7.5 mL
whole blood). Nonetheless, a recently reported study suggests that,
using this less stringent cut point, patients with positive findings do
have a modestly but statistically significant worse prognosis.
9
Recent studies have suggested that the CellSearch technique
may underestimate the number of EpCam-expressing cells. In this
regard, more efficient capture of these cells can be facilitated by use
of alternative solid-phase capture strategies, which are either based
on immune-capture that uses anti-EpCam or that exploits physical
characteristics that might distinguish cancer from normal hema-
topoietic cells, such as size or migration differences.
10
An alterna-
tive approach to enumeration of CTCs is to estimate their presence
by use of reverse transcriptase polymerase chain reaction to detect
epithelial transcripts, which presumably should not be present in
normal hematopoietic cells. This strategy has been applied in
breast, lung, prostate, and colon cancers,
11
and has been found to
be more sensitive than immune-capture techniques and to predict
prognosis in early-stage breast and colon cancers as well as in
metastatic prostate cancers.
12-14
Taken together, these studies suggest an exciting, but yet
unproven, potential for detection, enumeration, and monitoring
of CTCs in patients with a variety of solid tumors, either in the
early-stage or late-stage disease settings. However, none of the
assays are ideal; they lack both sensitivity and specificity. In partic-
ular, specificity may not be just technical, but biologic as well.
Technical nonspecificity implies that the assay detects an entity
that, after more careful scrutiny, is not found to be a cancer cell.
Perhaps more importantly, biologic nonspecificity implies that the
assay detects an entity that by all criteria is a cancer cell, but which
lacks the ability to invade, proliferate, and cause a metastasis.
Therefore, it would be of great value to not only enumerate CTCs
but to characterize them as well. In this regard, several authors have
reported the ability to analyze CTCs for a number of markers,
including among others HER2, insulin-like growth factor receptor-1,
urokinase-type plasminogen activator, BCL2, and the apoptotic
marker M30.
10
Cancer Stem Cells
It has long been appreciated that cancers are composed of heter-
ogeneous populations of cells. Evidence is rapidly accumulating that
many, if not most, cancers contain populations of cells that display
stem-cell properties.
15
These cancer stem cells (CSCs), by virtue of
their relative resistance to classical therapies, including radiation and
cytotoxic chemotherapy, may contribute to treatment failure and
relapse.
16,17
Furthermore, in preclinical models, CSCs have been dem-
onstrated to be the cells with the greatest invasive and metastatic
capacity.
18
If this is the case in patients with cancer, then one would
predict that CTCs may be enriched for CSCs compared with the
primary tumors from which they originated. Indeed, in women with
metastatic breast cancer, CTCs were found to be highly enriched for
cells that expressed the breast CSC markers CD44
⫹
/CD24
–19
or alde-
hyde dehydrogenase (ALDH1).
20
Interestingly, micrometastasis de-
tected in the bone marrow of these patients showed a similar CSC
profile, which suggests an important functional role of circulating
tumor stem cells (CTSCs) in mediating micrometastasis.
21
Although
micrometastases are enriched for cells expressing CSC markers, mac-
rometastases more closely resemble the profile of the primary tumor,
which suggests that micrometastases are initiated by cancer stem cells
that can then self-renew as well as generate bulk tumor populations at
distant sites.
CTSCs As Prognostic Biomarkers
If CTSCs are the cells that are responsible for the generation of
metastatic disease, then one would predict that assessment of these
cells for biologically important markers, such as those that confer
cell stemness, would provide more clinically relevant prognostic
and predictive information than simple enumeration—in other
words, biologic specificity. Indeed, one of the major limitations of
the CellSearch assay is the reliance of this technique on the expres-
sion of EpCam for cellular detection. Many CSCs display proper-
ties of epithelial mesenchymal transition (EMT) in which
expression of cell surface EpCam is downregulated.
22
The EpCam-
negative EMT-like state has been associated with increased meta-
static capacity in preclinical models and metastatic lesions in
patients.
23,24
These observations additionally support develop-
ment of methodology that is not dependent on EpCam expression,
not simply to increase sensitivity, but to provide the opportunity to
detect CTSCs that express the EMT phenotype—in other words, to
enhance biologic specificity.
Quantitative polymerase chain reaction (qPCR) might repre-
sent such a technique. However, a number of validated CSC mark-
ers, such as CD44 and ALDH, are also expressed in hematopoietic
stem cells, and therefore blind qPCR of whole blood will be fraught
with false-positive findings. To circumvent this problem, several
investigators have used negative selection with antibodies against
the hematopoietic-specific antigen CD45 followed by qPCR or cell
analysis on the remaining cell population using stem cell markers.
Although one must have significant concerns about the ability to
completely eliminate contamination of leukocytes with such a
strategy, this technique has been used to demonstrate an increased
proportion of CD44
⫹
or ALDH
⫹
CTCs in women with metastatic
breast cancer.
19,20
An alternative approach is the use of markers such as CD133.
CD133 has been reported to be expressed in a number of solid
tumor CSCs, but not by hematopoietic stem cells. In this issue of
the Journal of Clinical Oncology, Iinuma et al
25
report use of a
PCR-based assay to detect mRNAs for CD133 in combination with
those for cytokeratin (CK) and carcinoembryonic antigens (CEA)
in the CTCs of patients with colorectal cancer. The authors inves-
tigated this assay in a multi-institutional study involving 735 pa-
tients, including a training set of 420 patients from whom archived
specimens were retrospectively available and 315 patients who
were prospectively enrolled in a validation set. These investigators
report that the overall survival and disease-free survival of patients
with detectable CEA/CK/CD133
⫹
mRNA were significantly worse
than those of patients whose blood was negative for expression of
these markers (P⬍.003).
Importantly, the authors
25
report that the assay was not prog-
nostic in patients with Dukes’ stage A or Dukes’ stage B cancer who
were determined to have favorable prognoses on the basis of
clinical and pathologic features. The assay was prognostic in
patients with Dukes’ stage B cancer who were separated out
because of a less favorable prognosis on the basis of clinical and
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pathologic features (termed as separated patients in the study)
and in patients with Dukes’ stage C cancer. Clinically, these
findings are important. They suggest that patients with Dukes’
stage A and favorable Dukes’ stage B colorectal cancer have such
a low probability of recurrence that, even with this ultrasensi-
tive assay, one cannot find a group for whom adjuvant chemo-
therapy might be appropriate. In separated patients with Dukes’
stage B, none of whom received adjuvant chemotherapy, the
assay appears to identify a group of patients who, if chemother-
apy is effective, might benefit from it.
The results for Dukes’ stage C cancer raise a worrisome concern.
In addition to identifying a group of patients with poor prognosis in
the absence of therapy, does this assay also predict resistance to the
therapy itself? This hypothesis is completely plausible, given that a
major hallmark of cancer stem cellness is resistance to noxious stimuli,
such as chemotherapy.
16,17
One cannot answer this question without a
concurrent, randomly assigned, untreated control group, but the large
separation in the survival curves in patients with Dukes’ stage C can-
cer, all of whom received adjuvant chemotherapy, suggests that pa-
tients with circulating CEA/CK/CD133–positive cells may have
resistant disease. However, this concern is lessened by the observation
that the CSC marker CD133 seemed to be necessary to classify poor
prognosis in the untreated patients with Dukes’ stage B, but that the
CEA/CK assay was as prognostic as the CEA/CK/CD133 assay in the
group with Dukes’ stageC.
There are a number of limitations of this study, several of which
are acknowledged by the authors.
25
The use of the PCR assay does not
permit analysis at the single cell level, a feature that is important in CSC
analysis. Furthermore, the authors did not use negative selection to
remove hematopoietic cells. In fact, the authors acknowledge that
because CD133 is expressed in endothelial progenitor cells,
26
which
are known to be present in the circulation, they cannot conclusively
determine the cellular origin of CD133 mRNA. The development of
alternative strategies that permit isolation of individual CTSCs might
obviate some of these concerns. Approaches that use negative selec-
tion of hematopoietic cells followed by capture with CSC antigens
represent one such approach. Alternative approaches that are not
dependent on EpCam include cellular isolation on the basis of size or
microfluidic properties.
27
In conclusion, we are impressed by the rigorous technical and
clinical efforts the authors have applied to this prospectively
performed study of CTCs.
25
However, because of the limitations dis-
cussed above, and because therapy was applied according to standard
of care and not within a prospective randomized trial, these results
must be considered level of evidence II or III and not ready for use
when making clinical decisions.
28
The assay was not helpful in select-
ing patients with a favorable stage (Dukes’ stage A and favorable
Dukes’ stage B) who might benefit from therapy. One cannot deter-
mine whether separated patients with Dukes’ stage B with a positive
assay result will benefit from therapy, and likewise, from these results,
one cannot differentiate patients with Dukes’ stage C cancer who
might not have needed therapy.
Nevertheless, this study by Iinuma et al
25
does establish the
highly reproducible analytic validity of this assay in patients with
colorectal cancers. Moreover, the study raises fascinating hypoth-
eses that could and should be addressed in either archived speci-
mens from previously conducted trials or from ongoing or newly
designed randomized trials to determine the clinical utility of this
assay; it could have enormous utility for directing adjuvant therapy
for patients with this disease.
In addition, these results have implications for future clinical
trials of new therapeutic agents. To the extent that cancers follow a
stem cell model, current clinical response criteria may not accu-
rately predict patient outcome. The Response Evaluation Criteria
in Solid Tumors Group (RECIST) currently used to assess efficacy
in most clinical trials largely reflect the effects of therapeutic agents
on bulk tumor populations rather than the rarer CSC populations
that drive tumor growth and metastasis. In addition, pharmacody-
namic end points of targeted therapeutic agents are currently as-
sessed on bulk cell populations. This highlights the importance of
being able to assess the therapeutic effects of treatments on CSCs.
The utility of such an approach has been demonstrated in tissue
samples obtained before and after therapy in women undergoing
neoadjuvant therapy for locally advanced breast cancer.
16
How-
ever, the application of such an approach is limited by the technical
difficulty of obtaining pre- and post- treatment biopsies from
patients with metastatic solid malignancies. The ability to readily
obtain samples of CTSCs may obviate these difficulties and provide
the equivalent of multiple serial biopsies. In addition, the develop-
ment of technologies capable of reliably isolating and molecularly
characterizing CTSCs should facilitate the development of CSC-
targeted therapeutics.
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Although all authors completed the disclosure declaration, the following
author(s) indicated a financial or other interest that is relevant to the subject
matter under consideration in this article. Certain relationships marked
with a “U” are those for which no compensation was received; those
relationships marked with a “C” were compensated. For a detailed
description of the disclosure categories, or for more information about
ASCO’s conflict of interest policy, please refer to the Author Disclosure
Declaration and the Disclosures of Potential Conflicts of Interest section in
Information for Contributors.
Employment or Leadership Position: None Consultant or Advisory
Role: Max S. Wicha, OncoMed Pharmaceuticals (C), Pfizer (C), Dompe
(C); Daniel F. Hayes, OncoMed Pharmaceuticals (C), Pfizer (C), Dompe
(U) Stock Ownership: Max S. Wicha, OncoMed Pharmaceuticals
Honoraria: None Research Funding: Max S. Wicha, Dompe; Daniel F.
Hayes, Veridex, Pfizer, Novartis Expert Testimony: None Other
Remuneration: None
AUTHOR CONTRIBUTIONS
Manuscript writing: All authors
Final approval of manuscript: All authors
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■■■
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