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January 2005
Cover Story
William Check, PhD
In Fyodor Dostoyevsky’s classic novel The Brothers
Karamazov, there is a point at which Ivan Karamazov, suffering from
“brain fever,” hallucinates that he is conversing with the devil in the
form of a down-at-heels gentleman. The gentleman-devil is complaining
of his ailments: “I’ve tried many doctors; they diagnose beautifully,
they have the whole of your disease at their fingertips, but they’ve no
idea how to cure you. There was an enthusiastic little student: ’You may
die,’ he said, ’but you’ll know what disease you are dying of!’”
Although medicine has advanced a long way in the 124 years since Dostoyevsky
wrote his novel, it is still plagued by the fact that diagnosis often
outruns therapeutics. This flaw particularly bedevils tumor marker assays,
including a recent version—detecting circulating tumor cells in
the blood. Being able to detect such cells would be very useful, says
Karen L. Kaul, MD, PhD, director of molecular diagnostics at Evanston
(Ill.) Northwestern Heathcare and professor of pathology and urology at
the Northwestern University Feinberg School of Medicine. “We could potentially
use it for screening, staging, prognosis, detecting relapse, monitoring
therapy, finding contamination of stem cell harvests, and evaluating lymph
nodes and surgical margins,” Dr. Kaul says. In her view, detecting circulating
tumor cells in the blood would be “a natural extension of what we do in
surgical pathology.”
Interest in detecting circulating tumor cells, or CTCs, grew strong this
summer with the publication of an article showing that breast cancer patients
with measurable metastatic disease who had CTCs had a significantly worse
prognosis (Cristofanilli M, et al. N Engl J Med. 2004;351:781-791).
Based on the data from this trial, the Food and Drug Administration cleared
the assay, called CellSearch Epithelial Cell Kit, used in the trial. “It’s
exciting to see these tools used in well-done clinical studies,” Dr. Kaul
says. Nonetheless, she believes this method is not yet ready for routine
clinical use. “Does showing correlation with outcome mean we are ready
to go live with these assays?” she asks. “Probably not. We don’t know
enough about, and the clinical relevance of, circulating tumor cells to
know what to tell patients about the results.”
Other molecular pathologists express similar views. “These results are
a step in the right direction,” says Wayne W. Grody, MD, PhD, professor
in the Divisions of Medical Genetics and Molecular Pathology in the Departments
of Pathology and Laboratory Medicine and of Pediatrics and Human Genetics,
UCLA School of Medicine. “I still would like to see more than one paper
demonstrating efficacy before we brought this into clinical use.”
One of the responses that Kevin C. Halling, MD, PhD, gets from clinicians
when he talks to them about this type of assay is, How are we going to
use the information? “Say a patient has circulating breast cancer cells,”
says Dr. Halling, co-director of the clinical molecular genetics laboratory,
Department of Laboratory Medicine and Pathology, Mayo Clinic. “Will you
be able to do something about it? Or is it just bad news for the patient?
The big question for assays that detect CTCs is whether they will change
clinical management. People who try to convince you this is a useful assay
will tell you that it helps make more-informed decisions about treatment
options. They may be right, but we need more studies to know for sure.”
More-informed decisions are precisely what Massimo Cristofanilli, MD,
who was first author on the article reporting results with the CellSearch
assay, says the test makes possible. “This test provides an extremely
important new prognostic factor,” says Dr. Cristofanilli, associate professor
in the Department of Breast Medical Oncology, University of Texas M.D.
Anderson Cancer Center. He is using the test now in all patients with
metastatic breast disease “to be more appropriate in my estimate of prognosis
and my treatment plan.” In his view, all women with metastatic breast
cancer about to start a new line of therapy should be offered the test.
“We already order tumor markers with much less clinical value,” he says,
“so why not use this test also?”
Daniel F. Hayes, MD, was also an investigator in the CellSearch trial.
Dr. Hayes, who is clinical director of the breast oncology program at
the University of Michigan Comprehensive Cancer Center, has worked for
many years on breast tumor markers and is one of the discoverers of CA
15-3. “I am, frankly, intensely proud of this trial,” says Dr. Hayes.
“Unlike almost any other tumor marker study, this was a prospective study
with carefully defined endpoints and followup times. So unlike most tumor
markers studies, it is very likely these data are correct.” Asked whether
the assay is appropriate for all women with metastatic breast cancer,
he answers, “With the qualifier ’every,’ the answer is ’no.’ I would use
it in selected patients.” Then he adds: “This test does not replace the
human brain. It doesn’t direct therapy, but it can help.”
Why such varied interpretations of the data? As usual with such a complex
issue, the devil is in the details.
Looking for the source of metastases in the bloodstream
reflects a view of cancer spread that derives from the “seed and soil”
hypothesis of 19th-century physician-scientist Steven Paget, who postulated
that tumor cells must be adapted to a particular tissue to land, take
root, and grow there. Contemporary science has verified that specific
genetic alterations are required for secondary growth, so that not all
tumor cells in the blood are capable of forming a metastasis, which is
one possible source of false-positive results.
It is also possible that the presence of tumor cells in the blood is
an “episodic” event, says Dr. Grody. “I have a sense that it is not a
constant, that you don’t always have tumor cells in the blood, even when
you have metastases,” he says. He speculates that, as a tumor grows, it
can break through a major vessel and spill “seeds.” Then it may be contained
again. “So that’s one reason why I think that circulating tumor cell assays
are not yet ready for prime time,” he says.
Another source of misleading results, Dr. Kaul says, is that “unfortunately,
most markers are tissue-specific, not tumor-specific.” PSA, tyrosinase,
cytokeratins, HER2, and mammaglobin are all examples. “Early research
fell on its face because of inadequate assays,” she says. “There were
a lot of poorly done studies that did not define sensitivity and specificity.”“
Dr. Kaul comes by her caution honestly—for the past decade she
has been following up a report on the value of molecularly identifying
circulating cancer cells to stage localized prostate cancer preoperatively,
a report that turned out to be, like Ivan Karamazov’s vision, less than
solid. “We got into this in 1994,” she says, “naively thinking we were
validating a test that was moving from research to clinical application
in short order. We are now in our fifth-generation assay and still in
research mode.”
In the original paper, RT-PCR was used to identify circulating cells
that made mRNA for PSA. The authors interpreted their data as showing
that patients who had such cells would have positive margins after radical
prostatectomy, and so shouldn’t have surgery, while those without circulating
cells would have negative margins and could be cured (Katz AE, et al.
Urology. 1994;43:765-775). “Patients jumped all over this,” Dr.
Kaul recalls. Many called urologists and asked how to get the test done.
“Patients believed they would no longer have to flip coins whether to
have surgery or go straight to radiation oncology,” Dr. Kaul says. One
of the urologists at Evanston Northwestern Healthcare came to her for
help in implementing an assay and validating these results. In their prospective
study, they found positive results about equally often in margin-negative
and margin-positive patients. “We concluded that at this level you couldn’t
molecularly stage prostate cancer,” Dr. Kaul says (Ignatoff JM, et al.
J Urol. 1997;158:1870-1874). Several other laboratories reached
the same conclusion. Subsequently, the original investigators were unable
to validate their results and softened their claims.
In the meantime, the test was licensed to a reference laboratory, which
later had to stop offering it, but not before many patients had the test
done. “I don’t know that we were doing a service” by offering the test
so quickly, Dr. Kaul says. “Patients could be devastated by a finding
of circulating tumor cells—and we don’t know its meaning.”
Dr. Hayes is well aware of the sins committed in the
tumor marker field. In what he calls his “diatribe” on this topic, he
says, “Tumor marker studies are often not done well. I’ve been responsible
for some of these myself.” One source of the problem: “FDA has much stricter
criteria for new drugs than new markers.” Tumor marker studies are often
retrospective, using archived sera—“whatever you’ve got in the freezer,”
as he puts it.
Dr. Hayes and his colleagues set out to design the CellSearch trial like
a new drug study, prospectively done with defined clinical and radiographic
examinations. The cutoff for positivity—5 CTCs/7.5 mL of blood—was
derived from a training set of 100 samples and verified in an independent
validation set of 77 samples. To reduce the effect of non-specificity
of the tumor marker used, CK19, immunomagnetic separation was done to
enrich for epithelial cells. Patients represented a broad cross-section
of those with metastatic disease about to start a new line of therapy,
ranging from women with new estrogen receptor-positive recurrences for
which they would get hormone therapy to those scheduled for third-line
chemotherapy, which might be their last treatment.
One significant finding was that the 50 percent of women who had elevated
CTCs at baseline (pre-therapy) had worse overall and disease-free survival.
“To me as a clinician, that is not so important,” Dr. Hayes says. “I don’t
think I am going to select initial therapy from knowing whether a woman
has a worse prognosis. Unfortunately, metastatic breast cancer patients
almost all die of their disease, so I am trying to choose a therapy most
likely to palliate that patient. And baseline CTCs don’t tell me which
is most likely to work.”
A more important finding clinically was that the CTC result at first
followup—three to four weeks after initiating therapy—was
equally prognostic. Dr. Hayes explains how he would use the followup CTC
value in practice. “Each time I see the patient after starting therapy
I ask, Are we accomplishing the goal of palliation—making the tumor
shrink with fewest side effects?” If the cancer is obviously progressing,
or the patient has intolerable toxicity, or the tumor is responding well,
his next step is clear. But in about half of patients the response to
therapy is unclear. Clinical examinations and radiography—bone scan,
CT, and MRI—sometimes clarify the situation. However, Dr. Hayes
says, scans are time-consuming and expensive. A patient can spend a day
in radiology. In addition, “Usually it doesn’t give you an indication
of where you are for two to three months. And although they are the gold
standard, radiologic scans are not terribly accurate. So after three cycles
I often find myself saying, ’Mrs. Jones, I just can’t figure out your
scans. Let’s go another three cycles and see what happens.’”
Classic tumor markers—CEA, CA 15-3, CA 27.29—are “reasonably
good,” he notes, at aiding decision-making. But very early in the patient’s
course they frequently go up before they go down. This tumor marker spike
or flare can last two to eight weeks. “So maybe 25 percent of the time
we see a false-positive spike,” Dr. Hayes says.
It is here that the CTC assay can help—in patients on their third
or fourth cycles of treatment with indeterminate responses by standard
evaluations in whom the clinician would like to try a few more cycles
of the same therapy. “If she has elevated CTCs, you are probably kidding
yourself and you should change therapy,” Dr. Hayes says. “You still have
to be a physician,” he emphasizes. “For patients with a clear clinical
course, you do what’s reasonable, regardless of what her cells are. But
if you are on the fence, you can use the test to help make the decision.
”We hoped that measuring CTCs would be as good as and cheaper than radiographs,“
Dr. Hayes continues. Unpublished data from the trial suggest that it may
be a better predictor of survival than classical measures, though Dr.
Hayes would like to see that tested prospectively. As for relative price,
a bone scan costs $700 to $800 and a CT scan $1,000 to $1,500. Quest Diagnostics,
which has licensed the test, charges $600. Of course, saving money requires
that physicians substitute the CTC assay for radiography, rather than
add it. But will they? ”We can’t answer that question at present,“ Dr.
Hayes says. ”Only time and data will tell.“
Dr. Cristofanilli points out other important outcomes of the trial. First,
CTCs were prognostic only for patients beginning chemotherapy, not those
on hormone therapy alone, possibly because response to hormone therapy
is slower. (Dr. Hayes points out that the assay was evaluated in various
subgroups, such as those starting a new hormone therapy, in unplanned
and retrospective analyses. The assay’s performance in such subgroups
should be prospectively tested, he says, in well-designed and properly
powered future clinical trials.) Second, the percentage of patients with
CTCs dropped from 49 percent at baseline to 28 percent at first followup,
correlating with the benefit of treatment. And those who went from positive
to negative CTCs at three to four weeks approached the prognosis of patients
without cells at baseline. ”So we could use this assay as a measure of
treatment efficacy in patients with metastatic disease,“ he says.
Clinically, Dr. Cristofanilli is using the baseline CTC result to influence
initial therapy for women with metastatic disease about to start a new
line of treatment. He gives two examples. If a patient with estrogen receptor-positive
disease has CTCs, he would probably start with chemotherapy rather than
hormone therapy. And for a patient with estrogen receptor-negative minimal
disease with no CTCs, he would use single-agent chemotherapy instead of
a combination.
Outside the university setting, in routine clinical practice, Dr. Cristofanilli
suggests, the CTC assay can be used to help patients and physicians make
good decisions. “Not necessarily treatment decisions,” he cautions, “because
investigations should come first.” That is, studies need to be done to
see whether basing treatment on the CTC assay will improve outcomes.
Dr. Hayes agrees: “What we don’t know in patients with CTCs is whether
changing therapy will do them any good. Are CTCs just detecting those
who are not going to respond to anything?” He suggests that the fact that
20 percent of patients converted from CTC-positive to CTC-negative by
first followup indicates that some patients with elevated cells respond
to therapy. But that doesn’t say whether therapy will help patients who
still have elevated cells at followup.
To address this question, Dr. Hayes and his colleagues in the Southwest
Oncology Group are designing a randomized trial in which they hope to
prospectively test the value of changing to an alternative chemotherapy
versus staying on the originally chosen first-line chemotherapy in patients
with metastatic disease. The precise trial design is not set, but they
hope to open this validation study this year.
CellSearch Epithelial Cell Kit is marketed by Veridex,
a subsidiary of Johnson & Johnson. “We think this is a major step forward
in being able to measure the biological activity of tumor cells,” says
Mark Myslinski, general manager of Veridex. “This can help the physician
and patient make a decision as to what course of treatment to pursue.”
The sample is preserved in a CellSave tube for up to 72 hours. To run
the assay, a laboratory must purchase AutoPrep, which prepares cells,
and CellSpotter Analyzer, which visualizes fluorescently tagged cells.
These instruments come from a separate company, Immunicon, and cost $120,000.
The automated system developed by Immunicon scientists uses antibody-coated
ferrous particles to separate EpCAM-expressing epithelial cells from whole
blood specimens. Confirmation that these are indeed epithelial cells is
provided by automated fluorescent microscopy after staining with fluorescently
labeled monoclonal antibodies against cytokeratins and CD45 (a pan-leukocyte
marker) as well as DAPI. A trained observer reviews computer-generated
composite images of the detected events. Cytokeratin-positive, DAPI-positive
events are tallied by the computer as epithelial cells and expressed as
“CTC/mL of blood collected” (usually 7.5 mL into a 10-mL Vacutainer),
while “events” that fail to stain for cytokeratin or DAPI, or that stain
for CD45, are not. A proprietary fixative has been identified that makes
it possible to mail samples to central laboratories for processing and
assay within 48 to 72 hours.
Quest Diagnostics decided to offer the assay because it wanted to “provide
patient care at the highest level possible,” says Gary Milburn, PhD, national
director of genomics and esoteric testing services at Quest, Chantilly,
Va. “We felt that this assay, having been FDA cleared for breast cancer
recurrence, gives us an opportunity to impact patient care.” Dr. Milburn
predicts clinicians will use it as a more sensitive alternative to traditional
followup, such as physical examination and bone scan. “Obviously more
studies need to be done to demonstrate what to do with patients who have
increased CTCs,” Dr. Milburn says. “But from my discussions with physicians,
they seem willing to react exactly as if a patient had an additional lump
or an indication from bone scan that there may be recurrence.” Dr. Milburn
heads a team of a dozen professionals whose job is to give talks at hospitals
to help educate physicians about Quest’s new tests.
Dr. Kaul takes issue with the interpretation of the FDA’s approval wording—“for
the enumeration of circulating tumor cells of epithelial origin . . .
in whole blood”—as a clinical indication. “It is no more clear how
to use it than tests that are not FDA approved,” she says. “It is excellent
that we are getting reagents in a standardized format. But until we have
more information about the clinical utility of these assays, I don’t think
we should be offering them for clinical care. Until we have proof that
patients should be treated differently based on CTCs, I don’t think we
should be using this assay for clinical decision-making.”
Dr. Kaul’s comments are consonant with those of the authors of the editorial
that accompanied the CellSearch article, Stephan Braun, MD, and Christian
Marth, MD, both of Innsbruck Medical University, Austria. They wrote:
“[A] cautionary note is warranted with respect to uncritical, immediate
adoption of this assay for routine use. . . . We still need to know, for
example, whether any change in the treatment based on the number of circulating
tumor cells alone will translate into a benefit in progression-free survival”
(N Engl J Med. 2004;351: 824-826). On the positive side, they
said the data “allow us to speculate that this assay will soon realize
its potential to change the standard of care for patients with metastatic
breast cancer.”
Other assays for CTCs are available for research use
only. One from Abbott/Vysis uses real-time PCR to look for mRNA for markers
of breast cancer plus β-2-microglobulin, an internal control. Abbott
holds intellectual property rights to the two breast-specific markers,
BU101 and BS106, as well as proprietary primers for CK19, which were designed
to avoid amplification of any of the identified pseudogenes, says Timothy
Stenzel, MD, PhD, medical director of Abbott Molecular Diagnostics, who,
before moving to Abbott, worked on these markers as part of a grant that
Abbott and Duke University received from the NCI-funded Early Detection
Research Network. In collaboration with Dr. Kaul, lymph nodes were tested
using BU101, CK19, and mammaglobin, another breast-specific marker. And
in collaboration with Lyndsay Harris, MD, of Dana Farber Cancer Institute,
the assays were used to detect CTCs in HER2-positive breast cancer patients.
Data have been submitted for publication.
“We are at a preliminary stage,” Dr. Stenzel says. “All our analytical
data are very good. Now the question is whether it has clinical utility.
That has not yet been established. We are looking to develop collaborations
with individuals who will help us show the clinical utility of our assays.
We hope to make our current assay available for clinical purposes in the
not-too-distant future.” Dr. Stenzel sees the first application as monitoring
response to therapy in advanced or metastatic patients.
Another application would be to see if detecting tumor cells in lymph
nodes as well as blood or bone marrow is a more accurate way of staging
than standard pathological methods. Dr. Stenzel notes that 20 percent
to 30 percent of node-negative women recur even though it looks like they
have been cured. “There must have been cancer cells remaining,” he says,
“which is the rationale for giving adjuvant chemotherapy to some node-negative
women.” Dr. Stenzel suggests that histological examination of lymph nodes
with H&E may not be sufficient to identify patients who will recur. “We
need to find out whether molecular detection will be more useful.”
ChromaVision offers a research-use-only kit for detecting CTCs using
immunomagnetic enrichment combined with an immunocytochemical assay for
CK8, 18, and 19. Visualization is done by image analysis with an automated
digital microscopy system and review by a pathologist, which introduces
a professional component. Early data have been published (Witzig TE, et
al. Clin Cancer Res. 2002;8:1085-1091).
In newly diagnosed breast cancer, cytokeratin 19-positive cells visualized
in bone marrow by immunohistochemistry are a highly prognostic finding,
according to Dr. Braun and his colleagues in Innsbruck. In a prospective
study with 552 patients, they showed that the presence of occult metastatic
cells in bone marrow of patients with stage I, II, or III disease was
an independent prognostic indicator of the risk of death from cancer (Braun
S, et al. N Engl J Med. 2000;342:525-533). Only one percent of
191 patients with nonmalignant disease had CK19-positive cells in bone
marrow, compared with 36 percent of breast cancer patients. Most important
for staging purposes, this finding was highly significant among 301 women
without lymph-node metastases at primary diagnosis: 14 of the 100 with
CK19-positive cells in bone marrow died of cancer-related causes, but
only two of the 201 without such cells. Unfortunately, it is difficult
to apply this finding, since bone marrow sampling is not a routine part
of the clinical workup of breast cancer patients.
For just over a year a qRT-PCR assay for CTCs in the blood of people
with colorectal cancer has been available from Targeted Diagnostics &
Therapeutics Inc., of West Chester, Pa., a company formed in 1994 specifically
to license the guanylyl cyclase C (GCC) marker from Thomas Jefferson University,
where it was discovered by Scott Waldman, MD, PhD, the Samuel M. V. Hamilton
professor of medicine and professor of biochemistry and molecular pharmacology
and director of the Division of Clinical Pharmacology. “GCC is very specific
for the colon,” says Daniel J. O’Shannessy, PhD, chief operating officer
of TDT. Colonic cancer cells continue to express the protein and its mRNA.
So any GCC-positive cells found in the blood automatically represent potential
metastatic disease. Dr. O’Shannessy estimates the sensitivity and specificity
of GCC as both greater than 95 percent and superior to that of CEA or
CK20, other commonly used markers for CRC. Because of GCC’s high specificity,
no enrichment step is necessary, according to Dr. O’Shannessy.
In a small (21 samples) retrospective study, GCC mRNA was expressed in
lymph nodes from all stage II colorectal cancer patients with recurrent
disease at five years but not in those from patients without recurrent
disease (Cagir B, et al. Ann Intern Med. 1999;131:805-812). Dr.
O’Shannessy notes that 35 to 40 percent of patients with Dukes B (nonmetastatic)
colorectal cancer have recurrence by three to five years and die of their
disease. Use of GCC may provide more definitive staging in these patients,
he suggests. Dr. Waldman has received a five-year grant from the National
Cancer Institute to compare staging with and without the GCC qRT-PCR assay.
The NCI has also awarded Dr. Waldman a five-year grant prospectively to
monitor therapy in 2,000 to 3,000 colorectal cancer patients using the
GCC blood test.
Looking ahead to future developments
Markers have been used not only to identify CTCs, but also to detect
free circulating nucleic acids—both DNA and RNA—in serum and
plasma, as well as sputum, stool, and nipple aspirates. “To me it’s more
of a curiosity,” says Dr. Kaul. “It amazes me that you can find it there,
particularly RNA.” However, free nucleic acid detection is an area of
active research and one that she predicts “we will be hearing more about.”
(Related article: Detecting free DNA)
Dr. Hayes sees improvement in the value of CTC assays coming from methods
that do “not just counting, but actually phenotype and genotype cells.”
He points to the “tail” on the survival curves of patients who had CTCs
in the CellSearch study. “Between 10 to 15 percent of patients who have
elevated cells still did well,” he says. “So they have a ’cancer’ cell
that may not be so bad to have.” Many researchers have been trying to
genotype and phenotype cells, not just circulating but also in the primary
tumor and in bone marrow, to find those that actually predict metastases.
Dr. Kaul also stresses the need for new markers to make these assays
more clinically useful. “Most markers we use now are rooted in decades-old
immunohistochemistry studies done in surgical pathology,” she says. In
particular, markers are needed that reflect progression, since “most tumor
cells in the circulation are probably not capable of forming metastases.”
As an example of one way to find new, more specific markers, she points
to work using serial analysis of gene expression to identify genes that
were expressed in breast cancer but absent in the expression profiles
of blood and bone marrow cells. Four markers were selected that identified
29 percent of samples from patients with breast cancer who had minimal
residual disease, compared with none in non-breast cancer patients (Bosma
AJ, et al. Clin Canc Res. 2002;8:1871-1877). The ultimate assay,
Dr. Kaul predicts, will use new multimarkers to identify cells and their
biological relevance.
William Check is a medical writer in Wilmette, Ill. |
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