What goes around, comes around:

Early in the course
of epithelial malignancy, tumor cells frequently can become detached
from a primary lesion and enter the peripheral blood stream.^2,3
These so-called “circulating tumor cells” (CTC), are the subject of a
surge in current exciting, ongoing investigation. Questions being
investigated include:

• As tumor cells gain access to the peripheral blood, do they
  invariably cause metastasis?
• What is the clinical implication of detectable or increasing
  numbers of CTCs?

Metastatic dissemination can be caused by the
hematogenous spread of malignant cells. This even includes, for example,
one exceedingly rare case report of donor derived prostate cancer in a
heart transplant recipient.⁴ As it turns out, the vast
majority of epithelial cells, when removed from their original
microenvironment, will undergo apoptosis. This is known as “anoikis”
(derived from Greek, meaning “homeless”), and it is estimated that the
half-life of CTCs is less than three hours.⁵ Literature
indicates that CTC enumeration is prognostically important and allows
oncologists to monitor patients for disease progression and malignancy
relapse. Furthermore, recent advances in detection methods may yield new
avenues for clinical applications.

Characterization of circulating tumor cells, in
many ways, is equivalent to a “liquid biopsy” which can be analyzed to
determine susceptibility to treatment regimens. In this way, the
selective pressure of chemotherapy can be observed as it forces tumor
cell evolution, giving an advantage to those cells which are
therapy-resistant. With longitudinal monitoring, it is possible to
serially analyze tumor biology and detect emergence of clonal
populations, which is an early indication of treatment failure.

What is the best way to find a needle in a haystack?

Interestingly, the first case of tumor cells
in the peripheral blood was reported in 1869.⁶ While
there has been monumental technological innovation since that time,
the challenge still exists of isolating cells on the order of single
digits in a background of billions of blood cells. Several methods
have proven effective for CTC detection; PCR⁷, EPISPOT⁸,
and microfilter enrichment⁹ will not be discussed
in detail here.

It is thought that immunocytochemistry is a more
elegant and clinically important approach. Broadly speaking, this
approach involves antibody-mediated capture or enrichment of CTCs,
followed by treatment with a nuclear stain
(4,6-diamidino-2-phenylindole, or DAPI), and two different fluorescent
antibodies — one with specificity for CD45, the other for cytokeratin.
Finally, visualization takes place via fluorescence microscopy or other
image analysis. Immunocytochemistry platforms include Cellsearch,
CTC-chip, and fiber-optic array scanning technology (FAST) (see Table
1):

“The best work in the pathology of cancer is done by those who
… are studying the nature of the seed.” —Stephen Paget¹

1. Cellsearch (Veridex, LLC)¹⁰ relies
on a clever immunomagnetic enrichment step. A patient sample is treated
with magnetic iron particles which are bound to antibodies specific for
epithelial-cell-adhesion-molecule (EpCAM). CTCs become coated with these
particles, and then target-cell enrichment takes place by employing a
magnetic field. Immunofluorescent antibodies are applied and images are
then taken in one focal plane where the CTCs and particles are located.
Cellsearch technology has been around for some years, and the majority
of the earlier immunocytometric research used this system. For that
reason, and due to promising results, Cellsearch is the only
CTC-analysis platform which currently has FDA approval.

2. The CTC-Chip is so named for a process which
employs a “chip” consisting of microscopic posts that are etched in
silicon and coated with antibodies specific for epithelial surface
antigens. Under carefully controlled microfluidic conditions, blood
samples are passed by this chip at flow rates as low as 1 mL/hour,
thereby decreasing shear stress and increasing the likelihood of
antigen/antibody interactions. Once epithelial CTCs are captured, they
are treated with fluorescent antibodies and DAPI, then visualized.

In one study,¹¹ CTCs were demonstrated
in 115 of 116 patient samples from a variety of epithelial malignancies,
including prostate, pancreatic, breast, colon, and non-small-cell lung
cancers. Samples from 20 healthy subjects were all negative for CTCs.
Between five and 1,281 CTCs/mL were demonstrated in these samples, and
in a small cohort of six patients, changes in the numeric measurements
of CTCs correlated well with changes in radiographically measured
primary-tumor diameter. Furthermore, it has been demonstrated that
molecular analysis can be accomplished using this approach. Cells from
patients with pulmonary and prostate adenocarcinoma have demonstrated
expression of thyroid transcription factor-1 and prostate-specific
antigen, respectively. This was done both via immunohistochemistry and
reverse transcription PCR. Last, cell membrane integrity was analyzed,
leading to an estimated viability of ~98% of captured cells.

3. FAST is a novel technique which
takes yet another approach.¹² It should be no surprise that
the primary strength of “FAST” is quickness. A patient sample is
incubated with immunofluorescent antibodies, allowing visualization of
epithelial derived cells. Due to more efficient scanning technology,
image analysis of 25 million cells takes only two minutes, which is 500
times faster than other automated digital-microscopic techniques. This
provides an advantage in the detection of rare events.

In one study,¹³ instead of using
antibodies for CTC enrichment, a patient sample was treated with
ammonium to cause red-cell lysis and then samples were resuspended and
applied to a glass slide where the remaining cells were fixed in place
with paraformaldehyde. Cell locations were recorded relative to known
markers via image-analysis software, and cells were then re-stained with
Wright-Giemsa. Since their locations did not change, these exceedingly
uncommon cells were found easily and viewed with bright field
microscopy. Interestingly, CTCs detected from a patient with lung
adenocarcinoma were morphologically similar to malignant cells
identified from the biopsy of her primary tumor, taken years before.

For a pathologist, the implications are quite
intriguing. Fluid analysis via light microscopy is the basis of
cytopathology. Perhaps, the same principles can be applied to yield
additional information in dealing with CTCs. This calls to mind the
cancer stem-cell theory,¹⁴ and begs the question: If only a
subset of the cells in a malignancy are tumorigenic, do they have a
distinct morphological appearance? If so, the prognostic value of CTC
analysis would increase significantly.

What does it all mean?

Once we
have gained some faith in the available and emerging CTC testing
methods, many new clinical avenues become available. Tumor cells can
enter the peripheral blood early and often, but we know that the
overwhelming majority of them will quickly undergo apoptosis. Some would
argue that CTCs are not necessarily tumorigenic and, therefore, are not
clinically useful. The following are some of the more enticing
applications of CTC analysis, and this is, by no means, an exhaustive
list.

1. CTC enumeration is prognostic and
predictive. In a prospective trial of nearly 200 patients with
metastatic breast cancer,19 greater numbers of CTCs, measured via
Cellsearch, were independently found to be unfavorable and associated
with a shorter mean time to progression and a shorter mean overall
survival. At three to four weeks following initiation of chemotherapy,
greater circulating CTCs were once again associated with shorter mean
time to progression and mean overall survival. Additionally, this method
“gave a reliable estimate of disease progression and survival earlier
than estimations made with the use of traditional imaging methods (three
to four weeks vs. eight to 12 weeks after the initiation of therapy,
respectively).”¹⁹

The data show that the presence of greater than
five CTCs in patients is prognostic of worse outcome and also predictive
of poor response to therapy at three to four weeks. The threshold of
five CTCs per 7.5 mL of blood was determined via a training set of
patient samples and “most clearly distinguished patients with rapid
progression of disease from those with slow progression.”¹⁹

Similar trials have been conducted in patients
with many epithelial malignancies, including breast cancer,^20,21
prostate cancer,^7,2,23 colon cancer,^6,24 and renal
cell carinoma.²⁵ Additionally, RT-PCR has been used for CTC
detection in melanoma patients, and higher levels are shown to correlate
with shorter progression-free survival and overall survival.^{26, 27}
This body of research demonstrates not only the predictive and
prognostic value of CTC detection but also its usefulness in detecting
relapse and the correlation between greater CTCs and higher cancer
stage.

2. CTC analysis allows for detection of
emerging tumor clones.¹⁸
There is a subset of patients with non-small-cell lung cancers who
harbor an activating epidermal growth-factor receptor mutation. These
patients respond profoundly to tyrosine kinase inhibitor therapy, but a
further mutation (T790M) confers a survival advantage to the tumor
cells. One study has shown that when this drug-resistance mutation was
present in primary tumor biopsies, prior to initiation of systemic
therapy, the mean progression-free survival was 7.7 months, compared
with 16.5 months if the mutation was not present.

Upon analysis of DNA extracted from CTCs, in the
above patient population, a greater number of amplification cycles was
required for detection early on, indicating the presence of less nucleic
acid and fewer cells harboring this mutation. Later in the course of
these patients’ disease, fewer amplification cycles were required,
meaning that greater numbers of cells with this mutation are present,
and it is important to note that this correlated with clinical relapse.

These results imply that a clonal tumor evolution
is being witnessed under the selective pressure of chemotherapy. It
follows that the next step is to use this information to guide
management. With serial CTC analysis, the emerging clonal expansion
which precedes upcoming relapse can be objectively measured. Future
studies will determine if a change in therapy can be dictated sooner via
CTC detection of these emerging subpopulations rather than waiting for
clinical or radiographic progression.

3. CTC analysis may reveal differences
between the circulating cells and primary lesion. How should we
treat patients when there is a discrepancy in comparing the primary
lesion and the CTCs? One study shows breast-cancer patients with
HER2-positive disseminated tumor cells, and a HER2-negative primary
lesion.²⁸
Disseminated tumor cells in this case were isolated from bone-marrow
aspirates, rather than peripheral blood, but it may be speculated that
CTCs would also demonstrate HER2 positivity. Would these patients
benefit from trastuzumab, targeted therapy that is reserved for patients
with HER2 positivity in the primary cancer?

4. Can CTC detection compete with
conventional assays and radiography for cancer-patient monitoring?
The answer is yes according to the following research. In one study of
castration-resistant prostate-cancer patients, “CTC counts predicted
overall survival better than prostate specific antigen decrement
algorithms at all time points.”²³ Another study of metastatic
breast-cancer patients showed advantages of CTC enumeration relative to
radiology for disease monitoring.²⁹ Based on the presence or
absence of radiographic progression, and also based on whether patients
had: >=5 CTCs per 7.5 mL of blood, they can be arranged into four groups
(see Table 2).

It is not surprising that patients with greater
numbers of CTCs — in addition to radiographic evidence of worsening
disease — have the most dismal outcome. Conversely, fewer CTCs and lack
of radiographic progression confers longer survival. But when we compare
patients who have just one of these two findings (either greater numbers
of CTCs or radiographic progression), we find that CTCs are the more
important marker for disease status.

As with any emerging technology, the prospects
for application appear endless, and each question answered seems to
raise more. For example:

• Can CTC detection find occult malignancy?
• What is the utility of this technology in establishing a primary
  diagnosis?
• Is there any role in primary cancer screening?
• What does enumeration of CTCs reveal about tumor biology?
• Does a greater number indicate vascularity or predisposition to
  invasion?
• Will this help determine whether or not patients with operable
  cancer require adjuvant chemotherapy?

Current clinical research

This field remains largely untapped in
clinical settings. Newer CTC assays have not yet gained enough
clinical inertia and, subsequently, many physicians still do not
consider them to be a part of the oncologist’s armament. But
Cellsearch has been approved by the FDA, and there is currently a
trial underway designed to establish the usefulness of CTC
enumeration.

The presence of CTC in peripheral blood during or
after adjuvant therapy is associated with poor prognosis,³⁰
and so it could be argued that their presence during treatment is reason
for patients to switch to different therapy. S0500 by the The Southwest
Oncology Group addresses this issue. The study is designed to record the
number of CTCs for breast-cancer patients who are about to begin
systemic therapy. Three weeks later, CTCs again will be counted. For
patients with =5 CTC in 7.5 mL of blood,
however, who still have this number after three weeks of chemo, will be
considered equivalent to treatment failure. Patients in this so-called
treatment-failure group will then be randomized into two arms as
follows: 1) Either they will continue with therapy and be monitored for
clinical or radiographic progression per standard protocol, or 2) they
will discontinue the apparently ineffective chemoregimen in favor of
some alternative, which may prove beneficial.³¹

Currently, the American Society of Clinical
Oncology feels that there is insufficient evidence to support routine
CTC monitoring for clinical practice.³² This field is rapidly
evolving, however, and new techniques are being developed. Ongoing
trials demonstrate that CTCs can be enumerated and characterized to
provide prognostic and predictive information. Tumor-cell analysis
allows for detection of therapeutic targets. Additionally, longitudinal
evaluation can reveal the evolution and clonal selection of tumor
populations with resistance to chemotherapy. The future of CTC assays in
clinical practice awaits more sensitive platforms (such as CTC-Chip and
FAST) to become standardized and available for widespread use in
laboratories. In the near future, this technology may guide further
targeted therapy and, thereby, improve patient outcomes.

Thomas Gage, MD, is a resident in
Pathology at Beth Israel Deaconess Medical Center in Boston, MA; and
Shu-Ling Fan, PhD,
D(ABCC), F(ACB), is an instructor in Pathology at Harvard
Medical School and assistant director of Clinical Chemistry in the
Department of Pathology at Beth Israel Deaconess Medical Center.

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