Note: Descriptions are shown in the official language in which they were submitted.
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
METHODS AND REAGENTS FOR THE RAPID AND EFFICIENT
ISOLATION OF CIRCULATING CANCER CELLS
FIELD OF THE INVENTION
This invention relates to the fields of oncology
and diagnostic testing. The invention is useful for
cancer screening, staging, monitoring for chemotherapy
treatment responses, cancer recurrence or the like.
More specifically, the present invention provides
reagents, methods and test kits which facilitate
analysis and enumeration of tumor cells, or other rare
cells isolated from biological samples.
HACKGROITND OF THE INVENTION
Each year in the United States, approximately
600,000 new cases of cancer are diagnosed; one out of
every five people in this country will die from cancer
or from complications associated with its treatment.
Considerable efforts are continually directed at
improving treatment and diagnosis of this disease.
Most cancer patients are not killed by their
primary tumor. They succumb instead to metastases:
multiple widespread tumor colonies established by
malignant cells that detach themselves from the original
tumor and travel through the body, often to distant
sites. If a primary tumor is detected early enough, it
can often be eliminated by surgery, radiation, or
chemotherapy or some combination of those treatments.
Unfortunately, the metastatic colonies are harder to
1
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
detect and eliminate and it is often impossible to treat
all of them successfully. Therefore, from a clinical
point of view, metastasis can be considered the
conclusive event in the natural progression of cancer.
Moreover, the ability to metastasize is the property
that uniquely characterizes a malignant tumor.
Cancer metastasis comprises a complex series of
sequential events. These are: 1) extension from the
primary locus into surrounding tissues; 2) penetration
into body cavities and vessels; 3) release of tumor
cells for transport through the circulatory system to
distant sites; 4) reinvasion of tissue at the site of
arrest; and 5) adaptation to the new environment so as
to promote tumor cell survival, vascularization and
tumor growth.
Based on the complexity of cancer and cancer
metastasis and the frustration in treating cancer
patients over the years, many attempts have been made to
develop diagnostic tests to guide treatment and monitor
the effects of such treatment on metastasis or relapse.
Such tests presumably could also be used for cancer
screening, replacing relatively crude tests such as
mammography for breast tumors or digital rectal exams
for prostate cancers. Towards that goal, a number of
tests have been developed over the last 20 years and
their benefits evaluated. One of the first attempts was
the formulation of an immunoassay for carcinoembryonic
antigen [CEA]. This antigen appears on fetal cells and
reappears on tumor cells in certain cancers. Extensive
efforts have been made to evaluate the usefulness of
testing for CEA as well as many other "tumor" antigens,
such as PSA, CA 15.3, CA125, PSMA, CA27.29. These
2
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
efforts have proven to be somewhat futile as the
appearance of such antigens in blood have not been
generally predictive and are often detected when there
is little hope for the patient. In the last few years,
however, one test has proven to be useful in the early
detection of cancer, vi.z., Prostate Specific Antigen
[PSA] for prostate cancers. When used with follow-up
physical examination and biopsy, the PSA test has played
a remarkable role in detecting prostate cancer early, at
the time when it is best treated.
Despite the success of PSA testing, the test leaves
much to be desired. For example, high levels of PSA do
not always correlate with cancer nor do they appear to
be an indication of the metastatic potential of the
tumor. This may be due in part to the fact that PSA is
a component of normal prostate tissue as well as other
unknown factors. Moreover, it is becoming clear that a
large percentage of prostate cancer patients will
continue to have localized disease which is not life
threatening. Based on the desire to obtain better
concordance between those patients with cancers that
will metastasize and those that won't, attempts have
been made to determine whether or not prostate cells are
in the circulation. When added to high PSA levels and
biopsy data, the existence of circulating tumor cells
might give indications as to how vigorously the patient
should be treated.
The approach for determining the presence of
circulating prostate tumor cells has been to test for
the expression of messenger RNA of PSA in blood. This
is being done through the laborious procedure of
isolating all of the mRNA from a blood sample and
3
CA 02432363 2003-07-03
WO 99141613 PCTlUS99103073
performing reverse transcriptase PCR. As of this date,
(Gomella LG. J of Urology. 158:326-337(1997)) no good
correlation exists between the presence of such cells in
blood and the ability to predict which patients are in
need of vigorous treatment. It is noteworthy that PCR
is difficult, if not impossible in many situations, to
perform quantitatively, i.e., determine number of tumor
cells per unit volume of biological sample.
Additionally false positives are often observed using
this technique. There is an added drawback which is
that there is a finite and practical limit to the
sensitivity of this technique based on the sample size
examined. Typically, the test is performed on l05 to 106
cells purified away from interfering red blood cells.
This corresponds to a practical lower limit of
sensitivity of one tumor cell/ 0.1 ml of blood. Hence,
there needs to be about 10 tumor cells in a ml of blood
before signal is detectable. As a further
consideration, tumor cells are often genetically
unstable. Accordingly, cancer cells having genetic
rearrangements and sequence changes may be missed in a
PCR assay as the requisite sequence complementarity
between PCR primers and target sequences can be lost.
In summary, a useful diagnostic test needs to be
very sensitive and reliably quantitative. If a blood
test can be developed where the presence of a single
tumor cell can be detected in one ml of blood, that
would correspond on average to 3000 - 4000 total cells
in circulation. In innoculum studies for establishing
tumors in animals, that number of cells can indeed lead
to the establishment of a tumor. Further if 3000-4000
circulating cells represents 0.01% of the total cells in
4
.~........~....._.......__... ......____ _. _.._ _ ...... ..__ ____ -
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
a tumor, then it would contain about 4 x 10' total cells.
A tumor containing that number of cells would not be
visible by any technique currently in existence. Hence,
if tumor cells are shed in the early stages of cancer, a
test with the sensitivity mentioned above would detect
the cancer. If tumor cells are shed in some functional
relationship with tumor size, then a quantitative test
would be beneficial to assessing tumor burden.
Heretofore there has been no information regarding the
existence of circulating tumor cells in very early
cancers. Further, there are very considerable doubts in
the medical literature regarding the existence of such
cells and the potential of such information. The
general view is that tumors are initially well confined
and hence there will be few if any circulating cells in
early stages of disease. Also, there are doubts that the
ability to detect cancer cells early on will give any
useful information.
Based on the above, it is apparent that a method
for identifying those cells in circulation with
metastatic potential prior to establishment of a
secondary tumor is highly desirable, particularly early
on in the cancer. To appreciate the advantage such a
test would have over conventional immunoassays, consider
that a highly sensitive immunoassay has a lower limit of
functional sensitivity of 10-I'moles. If one tumor cell
can be captured from a ml of blood and analyzed, the
number of moles of surface receptor, assuming 100,000
receptors per cell would be 10"19 moles. Since about 300
molecules can be detected on a cell such an assay would
have a functional sensitivity on the order of 10-ZZ
moles, which is quite remarkable. To achieve that level
5
CA 02432363 2003-07-03
WO 99l41613 PCT/US99/03073
of sensitivity in the isolation of such rare cells, and
to isolate them in a fashion which does not compromise
or interfere with their characterization is a formidable
task.
Many laboratory and clinical procedures employ bio-
specific affinity reactions for isolating rare cells
from biological samples. Such reactions are commonly
employed in diagnostic testing, or for the separation of
a wide range of target substances, especially biological
entities such as cells, proteins, bacteria, viruses,
nucleic acid sequences, and the like.
Various methods are available for analyzing or
separating the above-mentioned target substances based
upon complex formation between the substance of interest
and another substance to which the target substance
specifically binds. Separation of complexes from
unbound material may be accomplished gravitationally,
e.g. by settling, or, alternatively, by centrifugation
of finely divided particles or beads coupled to the
target substance. If desired, such particles or beads
may be made magnetic to facilitate the bound/free
separation step. Magnetic particles are well known in
the art, as is their use in immune and other bio-
specific affinity reactions. See, for example, US
Patent No. 4,554,088 and Immunoassays for Clinical
Chemistry, pp. 147-162, Hunter et al. eds., Churchill
Livingston, Edinburgh (1983). Generally, any material
which facilitates magnetic or gravitational separation
may be employed for this purpose. However, it has
become clear that magnetic separation means are the
method of choice.
Magnetic particles can be classified on the basis
6
_..,~....,....~. _..... _..... ___....,_.,_.~__ ____.
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
of size as large (1.5 to about 50 microns), small (0.7-
1.5 microns), or colloidal (<200nm), which are also
referred to as nanoparticles. The latter, which are
also known as ferrofluids or ferrofluid-like materials
and have many of the properties of classical
ferrofluids, are sometimes referred to herein as
colloidal, superparamagnetic particles.
Small magnetic particles of the type described
above are quite useful in analyses involving bio-
specific affinity reactions, as they are conveniently
coated with biofunctional polymers (e.g., proteins),
provide very high surface areas and give reasonable
reaction kinetics. Magnetic particles ranging from 0.7-
1.5 microns have been described in the patent
literature, including, by way of example, US Patent Nos.
3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773;
4,554,088; and 4,659,678. Certain of these particles
are disclosed to be useful solid supports for
immunological reagents.
Small magnetic particles, such as those mentioned
above, generally fall into two broad categories. The
first category includes particles that are permanently
magnetizable, or ferromagnetic; and the second comprises
particles that exhibit bulk magnetic behavior only when
subjected to a magnetic field. The latter are referred
to as magnetically responsive particles. Materials
displaying magnetically responsive behavior are
sometimes described as superparamagnetic. However,
materials normally considered ferromagnetic, e.g.,
magnetic iron oxide, may be characterized as
superparamagnetic when provided in crystals of about
30nm or less in diameter. Relatively larger crystals of
7
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
ferromagnetic materials, by contrast, retain permanent
magnet characteristics after exposure to a magnetic
field and tend to aggregate thereafter due to strong
particle-particle interactions.
Like the small magnetic particles mentioned above,
large magnetic particles (> 1.5 microns to about 50
microns) can also exhibit superparamagnetic behavior.
Typical of such materials are those described by
Ugelstad in US Patent No.4,654267 and manufactured by
Dynal, (Oslo, Norway). The Ugelstad process involves
the synthesis of polymer particles which are caused to
swell and magnetite crystals are embedded in the swelled
particles. Other materials in the same size range are
prepared by synthesizing the polymer particle in the
presence of dispersed magnetite crystals. This results
in the trapping of magnetite crystals in a polymer
matrix, thus making the resultant materials magnetic.
In both cases, the resultant particles have
superparamagnetic behavior, which is manifested by the
ability to disperse readily upon removal of the magnetic
field. Unlike magnetic colloids or nanoparticles
previously referred to and discussed in further detail
below, these materials, as well as small magnetic
particles, are readily separated with simple laboratory
magnetics because of the mass of magnetic material per
particle. Thus, separations are effected in gradients
from as low as a few hundred gauss/cm on up to about 1.5
kilogauss/cm. Colloidal. magnetic particles, (below
approximately 200nm),on the other hand, require
substantially higher magnetic gradients because of their
diffusion energy, small magnetic mass per particle and
Stokes drag.
8
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
US Patent No. 4,795,698 to Owen et al. relates to
polymer-coated, colloidal, superparamagnetic particles
which are produced by the formation of magnetite from
Fe''/Fe`3 salts in the presence of polymer. US Pat. No.
4,452,773 to Molday describes a material similar in
properties to those described in Owen et al., which is
produced by forming magnetite and other iron oxides from
Fe'2/Fei3 via base addition in the presence of very high
concentrations of dextran. The resulting particles from
both procedures exhibit an appreciable tendency not to
settle from aqueous suspensions for observation periods
as long as several months. Materials so produced have
colloidal properties and have proved to be very useful
in cell separation. The Molday technology has been
commercialized by Miltenyi Biotec, Bergisch Gladbach,
Germany and Terry Thomas, Vancouver, Canada.
Another method for producing superparamagnetic,
colloidal particles is described in US Pat. No.
5,597,531. In contrast to the particles described in
the Owen et al., or Molday patents, these latter
particles are produced by directly coating a
biofunctional polymer onto pre-formed superparamagnetic
crystals which have been dispersed by high power sonic
energy into quasi-stable crystalline clusters ranging
from 25 to 120nm. The resulting particles, referred to
herein as direct-coated particles, exhibit a
significantly larger magnetic moment than colloidal
particles of the same overall size, such as those
described by Molday or Owen et al.
Magnetic separation techniques are known wherein a
magnetic field is applied to a fluid medium in order to
separate ferromagnetic bodies from the fluid medium. In
9
_..,..,..~.,~.~ ...,._..._..-._.,..._ ___
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
contrast, the tendency of colloidal, superparamagnetic
particles to remain in suspension, in conjunction with
their relatively weak magnetic responsiveness, requires
the use of high-gradient magnetic separation (HGMS)
techniques in order to separate such particles from a
non-magnetic fluid medium in which they are suspended.
In HGMS systems, the gradient of the magnetic field,
i.e., the spatial derivative, exerts a greater influence
upon the behavior of the suspended particles than is
exerted by the strength of the field at a given point.
HGMS systems can be divided into two broad
categories. One such category includes magnetic
separation systems which employ a magnetic circuit that
is entirely situated externally to a separation chamber
or vessel. Examples of such external separators are
described in US Pat. No. 5,186,827 to Liberti et al. In
several of the embodiments described in this patent, the
requisite magnetic field gradient is produced by
positioning permanent magnets around the periphery of a
non-magnetic container such that the like poles of the
magnets are in a field-opposing configuration. The
extent of the magnetic field gradient within the test
medium that may be obtained in such a system is limited
by the strength of the magnets and the separation
distance between the magnets. Hence, there is a finite
limit to gradients that can be obtained with external
gradient systems.
Another type of HGMS separator utilizes a
ferromagnetic collection structure that is disposed
within the test medium in order to 1) intensify an
applied magnetic field and 2) produce a magnetic field
gradient within the test medium. In one known type of
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
internal HGMS system, fine steel wool or gauze is packed
within a column that is situated adjacent to a magnet.
The applied magnetic field is concentrated in the
vicinity of the steel wires so that suspended magnetic
particles will be attracted toward, and adhere to, the
surfaces of the wires. The gradient produced on such
wires is inversely propo:rtional to the wire diameter,
such that magnetic reach decreases with increasing
diameter. Hence, very high gradients can be generated.
One drawback of internal gradient systems is that
the use of steel wool, gauze material, or steel
microbeads, may entrap non-magnetic components of the
test medium by capillary action in the vicinity of
intersecting wires or within interstices between
intersecting wires. Var:ious coating procedures have
been applied to such internal gradient columns (see,
e.g., US Patent Nos. 5,693,539 to Miltenyi and 4,375,407
to Kronick), however, the large surface area in such
systems still creates recovery concerns due to
adsorption. Hence, internal gradient systems are not
desirable, particularly when recovery of very low
frequency captured entities is the goal of the
separation. Furthermore, they make automation difficult
and costly. Both the materials described by Owen et
al., and Molday require the use of such high gradient
columns.
In contrast, HGMS approaches using external
gradients for cell separation provide a number of
conveniences. Firstly, simple laboratory containers
such as test tubes, centrifuge tubes or even vacutainers
(used for blood collection) can be employed. When
external gradients are of the kind that produce
11
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
monolayers of separated cells, as is the case with
quadrupole/hexapole devices of the above-mentioned US
Pat. No.5,186,827 or the opposing dipole arrangement
described in US Patent 5,466,574 to Liberti et al.,
washing of cells or subsequent manipulations are
facilitated. Further, recoveries of cells from tubes or
similar containers is a simple and efficient process.
This is particularly the case when compared to
recoveries from high gradient columns. Such separation
vessels also provide another important feature, which is
the ability to reduce sample volume. For example, if a
particular human blood cell subset, (e.g. magnetically
labeled CD 34' cells), is isolated from a 10 ml blood
sample diluted 50% with buffer to reduce viscosity, a 15
ml conical test tube may be employed as the separation
vessel in an appropriate quadrupole magnetic device.
Starting with 15 mis of solution, a first separation is
performed, and the recovered cells are resuspended in 3
mis. A second wash/separation is then performed and the
isolated cells resuspended in a final volume of 200 ul.
After the washes and/or separations and resuspensions
to remove non-bound cells, CD 34' cells can effectively
be resuspended in a volunie of 200 l. When done
carefully in appropriately treated vessels using direct-
coated ferrofluids which have been optimized for these
separators, cell recovery is quite efficient in the 40-
90% range depending on antigen density. Such techniques
and reagents are essential to achieve the degree of
sensitivity required for the kinds of cancer testing
mentioned above.
The efficiency with which magnetic separations can
be done and the recovery and purity of magnetically
12
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
labeled cells will depend on many factors. These
include such considerations as the number of cells being
separated, the receptor density of such cells, the
magnetic load per cell, the non-specific binding (NSB)
of the magnetic material, the technique employed, the
nature of the vessel, the nature of the vessel surface,
the viscosity of the medium and the magnetic separation
device employed. If the level of non-specific binding
of a system is substantially constant, as is usually the
case, then as the target population decreases so will
the purity. As an example, a system with 0.8 % NSB that
recovers 80% of a population which is at 0'.25% in the
original mixture will have a purity of 25%. Whereas, if
the initial population was at 0.01% (one target cell in
106 bystander cells), and if the NSB were 0.001%, then
the purity would be 8%. The greater the purity, the
easier and better the analysis. Hence, it is clear that
extremely low non specific binding is required to
perform meaningful rare cell analysis.
Less obvious is the fact that the smaller the
population of a targeted cell, the more difficult it
will be to magnetically label and to recover.
Furthermore, labeling and recovery will markedly depend
on the nature of magnetic particle employed. For
example, when cells are incubated with large magnetic
particles, such as DynalTM beads, cells are labeled
through collisions created by mixing of the system, as
the beads are too large to diffuse effectively. Thus,
if a cell were present in a population at a frequency of
1 cell per ml of blood or even less, as may be the case
for tumor cells in very early cancers, then the
probability of labeling target cells will be related to
13
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
the number of magnetic particles added to the system and
the length of time of mixing. Since mixing of cells
with such particles for substantial periods of time
would be deleterious, it becomes necessary to increase
particle concentration as much a possible. There is,
however, a limit to the quantity of magnetic particle
that can be added, as one can substitute a rare cell
mixed in with other blood cells for a rare cell mixed in
with large quantities of magnetic particles upon
separation. The latter condition does not markedly
improve the ability to enumerate the cells of interest
or to examine them.
There is another drawback to the use of large
particles to isolate cells in rare frequencies (1 to 50
cells per ml of blood). Despite the fact that large
magnetic particles allow the use of external gradients
of very simple design and relatively low magnetic
gradient, large particles tend to cluster around cells
in a cage-like fashion making the cells difficult to see
or to analyze. Hence, the magnetic particles must be
released from the target cells before analysis, and
releasing the particles clearly introduces other
complications.
Based on the foregoing, high gradient magnetic
separation with an external field device employing
highly magnetic, low non-specific binding, colloidal
magnetic particles is the method of choice for
separating a cell subset of interest from a mixed
population of eukaryotic cells, particularly if the
subset of interest comprises but a small fraction of the
entire population. Such materials, because of their
diffusive properties, readily find and magnetically
14
._,~...m......,,.,...._._..~.~.,...__.__....__...~...._.__W_.._..._.,_-
..,__._...__a_.~...._..._
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
label rare events, such as tumor cells in blood. Such
separation generally relies upon the identification of
cell surface antigens that are unique to a specific cell
subset of interest, which in the case of tumor cells,
can be tumor antigens to which appropriate monoclonal
antibody conjugated ferrofluids can be targeted.
Alternatively, when examining a blood sample,
determinants on classes of cells such as epithelial
cells, which are normally not found in blood, can
provide an appropriate receptor.
There are other good reasons to employ a colloidal
magnetic material for such separations, providing an
appropriate magnetic loading can be achieved. With
appropriate loading, a sufficient force is exerted on a
cell such that isolation can be achieved even in a media
as viscous as that of moderately diluted whole blood.
As noted, colloidal magnetic materials below about 200
nanometers will exhibit Brownian motion which markedly
enhances their ability to collide with and magnetically
label rare cells. This is demonstrated in US Patent No.
5,541,072 where results of very efficient tumor cell
purging experiments are described employing colloidal
magnetic particles or ferrofluids having a mean diameter
of 100 nm. Just as importantly, colloidal materials
having a particle size at or below this size range do
not generally interfere with examination of cells.
Cells so retrieved can be examined by flow cytometry,
laser scanning microscopy, or by microscopy employing
visible or fluorescent techniques.
CA 02432363 2003-07-03
WO 99/41613 PCT/US99103073
SUMARY OF THE INVENTION
The present invention is based on several important
discoveries which have significant clinical
ramifications for the diagnosis and treatment of cancer.
These are 1) tumor cells are present in the blood of
patients considered to have clinically localized,
primary tumors; 2) the number of tumor cells present in
the circulation is correlatable with all stages of
cancer from its inception to its terminal stages; and 3)
changes in the number of tumor cells present in the
circulation is indicative of disease progression. A
decrease in the numbers of circulating tumor cells is
indicative of improvement in patient status or efficacy
of treatment, whereas an increase indicates a worsening
of the disease.
The present invention provides a rapid and
efficient screening method for the characterization of
not only tumor cells, but also rare cells, or other
biological entities from biological samples. The method
of the invention provides highly sensitive analytical
techniques which enable efficient enrichment for
entities of interest. This two stage methodology which
ensures enrichment of tar=get bioentities while
eliminating a substantial amount of debris and other
interfering substances prior to analysis, allows for
examination of sample sizes which would otherwise be
impractical. The method described herein combines
elements of immunomagnetic enrichment with
multiparameter flow cytometric, microscopic and
immunocytochemical analysis in a unique way. Other
means of enrichment such as density gradient
centrifugation or panning or alteration of target cell
16
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
density by appropriate labeling may also be utilized.
According to a preferred embodiment, the method of the
invention enables assaying whole blood for cancer
staging, monitoring and screening. The sensitive nature
of the assay facilitates the detection of residual
disease, thus making it possible to monitor for cancer
recurrence.
In one embodiment of the invention, a biological
specimen, which comprises a mixed cell population
suspected of containing the rare cell of interest is
obtained from a patient. An immunomagnetic sample is
then prepared by mixing the biological specimen with i.
magnetic particles which are coupled to a biospecific
ligand specifically reactive with a rare cell
determinant or a class of determinants different than
those found on blood cells, to the substantial exclusion
of other sample components, and ii. at least one
biospecific reagent which labels rare cells. The
resulting immunomagnetic sample is subjected to a
magnetic field which is effective to separate the sample
into an unlabeled fraction and a labeled, magnetic
fraction including the rare cell of interest, if any is
present in the specimen. The cell population so
isolated is then analyzed to determine the presence and
number of rare cells. In a preferred embodiment the
particles used in this method are colloidal
nanoparticles.
In another embodiment of the invention, a
biological specimen is obtained from a patient. An
immunomagnetic sample is then prepared wherein the
biological specimen is mixed with colloidal. magnetic
particles which have been coupled to a monoclonal
17
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
antibody reactive with the rare cell determinant or a
class of determinants different than those found on
blood cells. As an alternative to monoclonal
antibodies, single chain or engineered fragments of
antibodies may be employed. The preparation is subjected
to a magnetic field, enriching the rare cell component
of the specimen. A second set of monoclonal antibodies,
labeled with reporter molecules, are added to the sample
and the cells are again cnagnetically separated in order
to remove unbound reagent to lower background staining.
A nucleic acid dye or other reporter molecule capable of
identifying objects as cells, also referred to herein as
a cell specific dye, is added to the sample to allow
exclusion of any residual non-nucleated cells or other
sample components prior to analysis by flowcytometry,
microscopy, or other analytical platforms. Cell
specific dyes may be reactive with DNA, RNA, protein, or
lipids such that the amount of signal obtained is
typical for that obtained for cells or the image
obtained reveals typical features of a cell, such as
cell and nuclear membranes, nucleus, and mitochondria.
In a further embodiment of the invention, the
isolated cells are subjected to immunocytochemical
analysis by flowcytometry or other analytical platforms.
Such analysis facilitates diagnosis and provides
important information to the clinician.
The method of the invention may be used to
assess residual cancer cells in circulation following
medical, radiation, or surgical treatment to eradicate
the tumor. The method may be also be performed
periodically over a course of years to assess the
patient for the presence and number of tumor cells in
18
~~,~~,.,._.,_,,._..__..... ..__...,..._......_.___.._,__._...._.___...__.._.-.-
_.__~..
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
the circulation as an indicator of occurrence,
recurrence and/or progression of disease.
In yet another aspect of the present invention, a
coated, magnetic particle is provided which comprises a
nanoparticle core of magnetic material, and a base
coating material on the magnetic core in an amount
sufficient to hinder non.-specific binding of biological
macromolecules to the magnetic core. These magnetic
particles are characterized by extremely low non-
specific binding as well as highly efficient target
capture which are essential to achieve a level of
enrichment the enrichment required to effectively
isolate very rare cells. In an alternative embodiment,
a coated, magnetic particle is provided which comprises
the following: i. a nanoparticle core of magnetic
material; ii. a base coating material that forms a
discontinous coating on the magnetic core, providing at
least one area of discontinuity which, if accessible,
contributes to non-specific binding of the base coated
particle to biological macromolecules; and iii. an
additional coating material that hinders access to the
areas of discontinuity by biological macromolecules.
The magnetic core material of the particles described
immediately above may comprise at least one transition
metal oxide and a suitable base coating material
comprises a protein. Protein-s suitable for coating
magnetic particles include but are not limited to bovine
serum albumin and casein. The additional coating
material may be the original coating proteins or one
member of a specific binding pair which is coupled to
the base material on the magnetic core. Exemplary
specific binding pairs include biotin-streptavidin,
19
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
antigen-antibody, receptor-hormone, receptor-ligand,
agonist-antagonist, lectin-carbohydrate, Protein A-
antibody Fc, and avidin-biotin. In one embodiment, the
member of the specific binding pair is coupled to the
base coating material through a bifunctional linking
compound. Exemplary biofunctional linking compounds
include succinimidyl-propiono-dithiopyridine (SPDP), and
sulfosuccinimidil-4-[maleimidomethyl]cyclohexane-l-
carboxylate (SMCC), however a variety of other such
heterobifunctional linker compounds are available from
Pierce, Rockford, Ill.
The coated magnetic particles of the invention
preferably have between 70-90% magnetic mass. In a
preferred embodiment, a major portion of the magnetic
particles have a particle size in the range of 90-150
nm. Particles may be synthesized such that they are
more monodisperse, e.g., in the range of 90-120 nm or
120-150 nm. The particles of the invention are
typically suspended in a biologically compatible medium.
In a further aspect of the present invention, a
test kit is provided for screening a patient sample for
the presence of circulating rare cells. The screening
kit comprises:
i. coated, magnetic nanoparticles coupled, directly or
indirectly, to a biospecific ligand that has affinity
for a first characteristic determinant on a rare cell;
ii. at least one biospecific reagent having binding
specificity for a second characteristic determinant
present on a rare cell; and iii. a cell specific dye
for excluding other non-target or sample entities from
analysis.
In a particularly preferred embodiment, a kit is
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
provided for screening biological samples for
circulating cancer cells. The screening kit comprises:
i. coated, magnetic nanoparticles coupled, directly or
indirectly to a biospecific ligand that has affinity for
a first characteristic determinant on a cancer cell;
ii. at least one biospecific reagent having binding
specificity for a second characteristic determinant
present on a cancer cell; and iii. a cell specific dye
for excluding non-target entities from analysis. The
kits provided herein may further include an antibody
which has affinity for non-rare, or non-tumor cells, a
biological buffer, a permabilization buffer, a protocol
and, if desired, an information sheet. In a preferred
embodiment, the colloidal magnetic particles are
conjugated to anti-EpCAM (an antibody having binding
specificity for epithelial cell adhesion molecule), the
biospecific reagents comprise a panel of monoclonal
antibodies and the cell specific dye stains nucleic
acids.
The kits of the invention may contain reagents for
diagnosing the type of the metastatic cancer cells in
the circulation as well as the metastatic potential and
aggressiveness of such cells. In this embodiment the
kit contains the reagents recited above, yet also
comprises additional antibody markers to facilitate
cancer diagnosis. Using breast cancer as an example,
such antibodies may include anti-MUC-l, anti-estrogen
receptor, anti-progesterone receptor, anti-CA27.29,
anti-CA15.5, anti-cathepsin D, anti-p53, anti- urokinase
type plasminogen activator, anti-epidermal growth
factor, anti-epidermal growth factor receptor, anti-
BRCA1, anti-BRCA2, anti-prostate specific antigen, anti-
21
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
plasminogen activator inhibitor and/or anti-Her2-neu
antibodies. Additional markers for aggressiveness and
invasiveness are Lewis a (Lea), sialyl Lewis a(sLea),
the intergrins (CD49b, CD49c, CD29), gelatinase A and B
(MMP-2, MMP-9), tissue collagenase (MMP-1),fibroblast
activation protein (FAP), guanidinobenzoatase, CEA, S100
family (S100A4, mtsl, 18A2/mtsl, pEL-98, p9Ka,
metastasin), the Cyclins A and E, p27, p53, vascular
endothelilal growth factor (VGEF) and E-Cadherin.
In yet another embodiment of the invention, a test
kit is provided for monitoring a patient for recurrence
of the cancer, and/or response to therapy. This
particular kit may also be used to assess high risk
patients for the presence of particular tumor cells in
the blood. A kit suitable for monitoring a patient
would include containers, colloidal magnetic particles
conjugated to anti-EpCAM, at least one monoclonal
antibody specific for the particular cancer cells for
which the patient is being monitored and a fluorescent
reporter molecule which can identify the objects as
cells, such as nucleic acid or membrane dyes. A kit
suitable for monitoring breast cancer patients comprises
an antibody having binding affinity for a particular
breast cancer marker, for example Her-2-neu. The kits
described above are suitable for screening, diagnosing
and monitoring patients for breast cancer. It will be
appreciated by those skilled in the art that many
different cancers may be screened, diagnosed and
monitored according to the present invention simply by
varying the antibodies provided in the test kit. For
example, if a test subject were being assessed for the
presence of prostate cancer, antibodies specific for
22
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
prostate specific antigen may be employed. Other
markers for prostate cancer include prostatic acid
phosphatase, creatine kinase, thymosin b-15, p53, HPC1
basic prostate gene, and prostate specific membrane
antigen.
There is general agreement that the hallmark of
successful treatment of cancer is early diagnosis.
Based on the data presented herein, it appears that the
blood test of the present invention may be used to
screen the blood of patients who do not have a diagnosis
of cancer, in order to detect cancer earlier than is
possbile using other existing methods. Such patients
may include those with a family history of certain
cancers, patients with certain mutations known to be
associated with cancer, etc.
Since cancer cells invade surrounding tissue and
breakdown tissue barriers, we hypothesize that tumor
cells enter the tissue space and capillaries to
eventually end up in the blood very early in the
development of a solid tumor, i.e., when the tumor
contains 104-106 tumor cells. See Figure B. At that
point in time, the tumor cells undergo apoptotic cell
death, or become dormant because they are not yet able
to survive or grow, respectively, in an ectopic
environment. There are no techniques at present to
detect such small primary tumors. There are sensitive
techniques that are available for detecting certain
types of cancer when the tumors are larger. For
example, mammography can detect 2 x 108 breast cancer
cells at best. More often, tumors of the breast are
detected when there are between S x 108 to 109 tumor
cells. At this early stage, we hypothesize that most
23
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
shed tumor cells will die. However, during the many
generations that occur as a tumor grows between 106 to
10B-9 tumor cells, the genetically unstable clone of
tumor cells undergoes further genetic changes giving
rise to more rapidly growing and aggressive mutant
cells. It is very likely these cells that go on to
establish secondary tumors. However, in the majority of
tumors, the diagnosis is made very late, e.g., pancreas,
stomach, ovary, kidney, lung, colon, etc., are usually
diagnosed when there are 1010-1012 tumor cells. By this
time the tumor has frequently invaded surrounding
tissues and/or has metastasized. In light of the
foregoing, it is clear that any test which would
effectively detect circulating cancer cells prior to the
establishment of a secondary tumor would be extremely
beneficial in the diagnosis and treatment of cancer.
The blood test described herein enables such detection.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of model experiments in
which known number of tumor cells are spiked into
peripheral blood and retrieved after immunomagnetic
selection and analysis by either microscopy (Panel A) or
flowcytometry (Panel B).
Figure 2 shows flowcytometric analysis of cell
suspensions obtained after immunomagnetic cell selection
from 10 ml of blood from a patient having distant
metastasis of carcinoma of the breast, drawn 48, 175 and
300 days after this patient entered the study. After
immunomagnetic selection, the cells were stained with an
epithelial cell specific phycoerythrin (PE) conjugated
24
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
monoclonal antibody, a leukocyte specific CD45 PerCP
conjugated monoclonal antibody and a nucleic acid dye.
Events passing a threshold on the nucleic acid dye were
acquired into listmode and 85% of the sample was
analyzed. The tumor cells are highlighted and
illustrated in black and their number is shown in the
top right corner; the background events, consisting of
residual leukocytes and debris, are illustrated in gray.
Figure 3 shows epithelial cell number in 10 ml of
blood and clinical activity of the disease at different
time points for eight patients with activb carcinoma of
the breast. The clinical activity of the disease was
classified in categories 1 through 4, as set out in
Table 1. The bars at the top represent the length of
time of chemotherapy. Panel A, adriamycineTm (ADR) 90 and
110 mg/m2 respectively, Panel B, ADR 30 mg/m2/week,
VinorelbineTM (Vin) 20 mg/m2/week, ADR 160 mg, ADR 20
mg/m2/week, Panel C, vincristineTm (Vinc) 0.7 mg/m2/week,
metotrexate""'` (MTX) 30 mg/m2/week, Panel D, vinblastineTM
(Vinb) 7 mg/m2/week, ADR 20 mg/m2/week, Vinb 6 mg/m2/week,
5-fluoruracil (5FU) 700 mg/m2/week. Panel E, Vin 20
mg/m2/week; 5FU 800 mg/m2/week + Leukovorin"4 50 mg/m2/week.
Panel F, ifosfamide`'' (IF) 18 mg/m2/week; 5FU
850 mg/mZ/week + Leukovorin 35 mg/mZ/week, 5FU 605
mg/m2/week; Vin 20 mg/m2/week+ Leukovorin 30 mg/mZ/week.
Panel G, Vin 20 mg/m~/week, Panel H, Vin 20 mg/m2/week
Figures 4A-4D are a series of micrographs showing
the results obtained following analysis of
immunomagnetically selected cells from peripheral blood
of patients with a history of breast carcinoma. Panel
CA 02432363 2003-07-03
WO 99/41613 PCT/US99103073
A, cells from a patient three years after surgery
(T2N1M0) staining positive for cytokeratin. Panel B,
cell from a patient eight years after surgery (T2N1M1)
in complete remission stained with Wright Giemsa. Panel
C and D cells from a patient 2 years after surgery
(T2NOMO) stained with Wright Giemsa'. The images were
taken with a Pixera'rM digital camera with a 100X
objective.
Figures 5A-5C are a series of graphs showing the
correlation between severity of disease and circulating
epithelial cell number in three patients 0ith prostate
cancer.
Figure 6 is a graph which shows that circulating
epithelial cell number in patients with colon cancer is
significantly decreased after surgical removal of the
tumor.
Figure 7 is a graph which shows that circulating
epithelial cell number in patients with metastatic
disease of the colon increases with the severity and
extent of metastatic disease.
Figure 8 is a schematic diagram showing the
progression of cancer from a primary tumor to growing
metastases.
DETAILED DESCRIPTION OF THE INVENTION
According to a preferred embodiment, the present
invention provides compositions, methods and kits for
the rapid and efficient isolation of rare target
26
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
bioentities from biological samples. The methods
described may be used effectively to isolate and
characterize tumor cells present in a blood sample while
at the same time minimizing the selection of non-
specifically bound cells.
Many clinicians believe that cancer is an organ-
confined disease in its early stages. Based on the data
presented herein, it appears that this notion is
incorrect. Indeed, the data reveal that cancer is often
a systemic disease by the time it is first detected
using methods currently available. Hence, the presence
of tumor cells in the circulation can be used to screen
for cancer in place of, or in conjunction with, other
tests, such as mammography, or measurements of PSA. By
employing appropriate mononclonal antibodies directed to
specific markers on or in cells, or by using other
assays for cell protein expression, or by the analysis
of cellular mRNA, the organ origin of such cells may
readily be determined, e.g., breast, prostate, colon,
lung, ovarian or other non-hematopoietic cancers. Thus,
in cases where cancer cells can be detected, while there
are essentially no clinical signs of a tumor, it will be
possible to identify their presence as well as the organ
of origin. Because screening can be done with the
relatively simple blood test of the present invention
described herein, which functions with a high degree of
sensitivity, the test can be thought of as a "whole body
biopsy". Furthermore, based on the data set forth
herein, cancer should be thought of as a blood borne
disease characterized by the presence of potentially
very harmful metastatic cells, and therefore, treated
accordingly. In cases where there is absolutely no
27
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
detectable evidence of circulating tumor cells, e.g.,
following surgery, it may be possible to determine from
further clinical study whether follow-up treatment, such
as radiation or chemotherapy is required. Determining
the need not to treat, given the costs of such
therapies, is a significant and beneficial piece of
clinical information.
It is also clear from the present data that the
number of tumor cells in the ciruclation is related to
the stage of progression of the disease, from its
inception to the final phases of disease.
The term "target bioentities" as used herein refers
to a wide variety of materials of biological or medical
interest. Examples include hormones, proteins,
peptides, lectins, oligonucleotides, drugs, chemical
substances, nucleic acid molecules, (e.g., RNA and/or
DNA) and particulate analytes of biological origin,
which include bioparticles such as cells, viruses,
bacteria and the like. In a preferred embodiment of the
invention, rare cells, such as fetal cells in maternal
circulation, or circulating cancer cells may be
efficiently isolated from non-target cells and/or other
bioentities, using the compositions, methods and kits of
the present invention. The term "biological specimen"
includes, without limitation, cell-containing bodily,
fluids, peripheral blood, tissue homogenates, nipple
aspirates, and any other source of rare cells that is
obtainable from a human subject. An exemplary tissue
homogenate may be obtained from the sentinel node in a
breast cancer patient. The term "determinant", when
used in reference to any of the foregoing target
bioentities, may be specifically bound by a biospecific
28
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
ligand or a biospecific reagent, and refers to that
portion of the target bioentity involved in, and
responsible for, selective binding to a specific binding
substance, the presence of which is required for
selective binding to occur. In fundamental terms,
determinants are molecular contact regions on target
bioentities that are recognized by receptors in specific
binding pair reactions. The term "specific binding
pair" as used herein includes antigen-antibody,
receptor-hormone, receptor-ligand, agonist-antagonist,
lectin-carbohydrate, nucleic acid (RNA or DNA)
hybridizing sequences, Fc receptor or mouse IgG-protein
A, avidin-biotin, streptavidin-biotin and virus-receptor
interactions. Various other determinant-specific
binding substance combinations are contemplated for use
in practicing the methods of this invention, such as
will be apparent to those skilled in the art. The term
"antibody" as used herein, includes immunoglobulins,
monoclonal or polyclonal antibodies, immunoreactive
immunoglobulin fragments, and single chain antibodies.
Also contemplated for use in the invention are peptides,
oligonucleotides or a combination thereof which
specifically recognize determinants with specificity
similar to traditionally generated antibodies. The term
"detectably label" is used to herein to refer to any
substance whose detection or measurement, either
directly or indirectly, by physical or chemical means,
is indicative of the presence of the target bioentity in
the test sample. Representative examples of useful
detectable labels, include, but are not limited to the
following: molecules or ions directly or indirectly
detectable based on light absorbance, fluorescence,
29
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
reflectance, light scatter, phosphorescence, or
luminescence properties; molecules or ions detectable by
their radioactive properties; molecules or ions
detectable by their nuclear magnetic resonance or
paramagnetic properties. Included among the group of
molecules indirectly detectable based on light
absorbance or fluorescence, for example, are various
enzymes which cause appropriate substrates to convert,
e.g., from non-light absorbing to light absorbing
molecules, or from non-fluorescent to fluorescent
molecules. The phrase "to the substantial exclusion of"
refers to the specificity of the binding reaction
between the biospecific ligand or biospecific reagent
and its corresponding target determinant. Biospecific
ligands and reagents have specific binding activity for
their target determinant yet may also exhibit a low
level of non-specific binding to other sample
components. The term "early stage cancer" as used
herein refers to those cancers which have been
clinically determined to be organ-confined. Also
included are tumors too small to be detected by
conventional methods such as mammography for breast
cancer patients, or X-rays for lung cancer patients.
While mammography can detect tumors having approximately
2 x 108 cells, the methods of the present invention
should enable detection of circulating cancer cells from
tumors approximating this size or smaller. The term
"enrichment" as used herein refers to the enrichment of
mononuclear cells from a biological sample. In cases
where peripheral blood is used as the starting
materials, red cells are not counted when assessing the
extent of enrichment. Using the method of the present
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
invention, circulating epithelial cells may be enriched
relative to leucocytes to the extent of at least 2,500
fold, more preferably 5,000 fold and most preferably
10,000 fold. The preferred magnetic particles for use
in carrying out this invention are particles that behave
as colloids. Such particles are characterized by their
sub-micron particle size, which is generally less than
about 200 nanometers (nm) (0.20 microns), and their
stability to gravitational separation from solution for
extended periods of time. In addition to the many other
advantages, this size range makes them essentially
invisible to analytical techniques commonly applied to
cell analysis. Particles within the range of 90-150 nm
and having between 70-90% magnetic mass are contemplated
for use in the present invention. Suitable magnetic
particles are composed of a crystalline core of
superparamagnetic material surrounded by molecules which
are bonded, e.g., physically absorbed or covalently
attached, to the magneti.c core and which confer
stabilizing colloidal properties. The coating material
should preferably be applied in an amount effective to
prevent non specific interactions between biological
macromolecules_found in the sample and the magnetic
cores. Such biological macromolecules may include
sialic acid residues on the surface of non-target cells,
lectins, glyproteins and other membrane components. In
addition, the material should contain as much magnetic
mass/nanoparticle as possible. The size of the magnetic
crystals comprising the core is sufficiently small that
they do not contain a complete magnetic domain. The
size of the nanoparticles is sufficiently small such
that their Brownian energy exceeds their magnetic
31
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
moment. As a consequence, North Pole, South Pole
alignment and subsequent: mutual attraction/repulsion of
these colloidal magnetic particles does not appear to
occur even in moderately strong magnetic f:ields,
contributing to their solution stability. Finally, the
magnetic particles should be separable in high magnetic
gradient external field separators. That characteristic
facilitates sample handling and provides economic
advantages over the more complicated internal gradient
columns loaded with ferromagnetic beads or steel wool.
Magnetic particles having the above-described properties
can be prepared by modification of base materials
described in U.S. Patents Nos. 4,795,698, 5,597,531 and
5,698,271. Their preparation from those base materials
is described below.
Malignant tumors are characterized by their ability
to invade adjacent tissue. In general, tumors with a
diameter of 1 mm are vascularized and animal studies
show that as much as 4% of the cells present in the
tumor can be shed into the circulation in a 24 hour
period (Butler, TP & Gul.lino PM, 1975 Cancer Research
35:512-516). The shedding capacity of a tumor is most
likely dependent on the aggressiveness of the tumor.
Although tumor cells are shed into the circulation on a
continous basis, it is believed that none or only a
small fraction will give rise to distant metastasis
(Butler & Gullino, supra). Using the following
assumptions, one can approximate the frequency of tumor
cells in circulation as follows: 1. A tumor with a
diameter of 1 mm contains 10' cells, and 4% or 4 x 105
cells will be shed into the circulation in a 24 hour
period; 2. tumor cells only survive one circulatory
32
.~.,....~..~...~.....r.-.~,-............ ..__.d __ _
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
cycle; 3. a blood volume of about 5 liters; and 4. a
cardiac output of 5000 ml / minute. In such a case, the
frequency of tumor cells in peripheral blood of a
patient with a 1-mm diameter tumor is approximately 6
tumor cells / 100 ml of blood. Increase in tumor mass
might be expected to be proportional to an increase in
the frequency of the circulating tumor cells. If this
were found to be the case, methods available with this
level of sensitivity would facilitate both assessing
tumor load in patients with distant metastasis and also
assessing tumor load in patients with localized disease.
Detection of tumor cells in peripheral blood of patients
with localized disease has the potential not only to
detect a tumor at an earlier stage but also to provide
indications as to the potential invasiveness of the
tumor.
Several studies report the presence of carcinoma
cells in leukopheresis products harvested from patients
with carcinoma of the breast for autologous peripheral
blood stem cell transplantation (Brugger W, et al.
(1994) Blood 83:636-640; Brockstein BE, et al. (1996) J
of Hematotherapy 5:617; Ross AA, et al. (1993) Blood
82:2605; Ross AA. (1998) J of Hematotherapy. 7:9-18;
Moss TJ, et al. (1994) J. Hematotherapy. 3:163-163).
These findings prompted criticism of the use of this
procedure for autologous transplantation since the tumor
cells in the transplant product have the potential to
establish metastasis (Racila E, et al. (1998) PNAS USA.
95:4589-4594). Additionally, it was found that
leukopheresis products were more likely to contain tumor
cells when obtained from individuals with disseminated
disease (Brugger et al., 1994, supra). These studies,
33
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
however, do not report quantitative data, nor do they
report that tumor cells can be found in peripheral blood
of patients with localized disease. Given these
observations, one may. hypothesize that a highly
sensitive and quantitative test that counts the number
of tumor cells in peripheral blood may be used to
determine actual tumor load. To assess the feasibility
of such testing, a sensitive cellular assay was
developed which allows precise enumeration of
circulating carcinoma cells that is limited only by the
blood volume to be tested.
It should be noted that a number of different cell
analysis platforms can be used to identify and enumerate
the enriched samples. Examples of such analytical
platforms are Immunicon' s CellSpotterTM system, a
magnetic cell immobilizer for manual observation of
cells, and the CellTracksTM system, an automatic optical
scanning magnetic cell immobilizer described in US
patent numbers 5,876,593 and 5,985,153 respectively.
Both of these references disclose the respective
apparatus and methods for manual or automated
quantitative and qualitative cell analysis.
Other analysis platforms include Laserscanning
Cytometry (CompucyteTM), bright field base image analysis
(ChromavisionTM), and Capillary volumetry (Biometric
imagingTM )
The enumeration of circulating epithelial cells in
blood using the methods and compositions of a preferred
embodiment of the present invention is achieved by
immunomagnetic selection (enrichment) of epithelial
cells from blood followed by the analysis of the samples
34
CA 02432363 2003-07-03
WO 99/41613 PCTlUS99103073
by multiparameter flowcytometry. The immunomagnetic
sample preparation is important for reducing sample
volume and obtaining a 10 fold enrichment of the target
(epithelial) cells. The reagents used for the
multiparameter flowcytometric analysis are optimized
such that epithelial cells are located in a unique
position in the multidimensional space created by the
listmode acquisition of two lightscatter and three
fluorescence parameters. These include 1) an antibody
against the pan-leucocyte antigen, CD45 to identify
leucocytes (non-tumor cells); a cell type specific or
nucleic acid dye which allows exclusion of residual red
blood cells, platelets and other non-nucleated events;
and 3) a biospecific reagent or antibody directed
against cytokeratin or an antibody having specificity
for an EpCAM epitope which differs from that used to
immunomagnetically select the cells.
It will be recognized by those skilled in the art
that the method of analysis of the enriched tumor cell
population will depend on the intended use of the
invention. For example, in screening for cancers or
monitoring for recurrence of disease, as described
hereinbelow, the numbers of circulating epithelial cells
can be very low. Since there is some "normal" level of
epithelial cells, (very likely introduced during
venipuncture), a method of analysis which identifies
epithelial cells as normal or tumor cells is desirable.
In that case, microscopy based analyses may prove to be
the most accurate. Such examination might also include
examination of morphology, identification of known tumor
markers and or oncogenes. Alternatively, in disease
states wherein the number of circulating epithelial
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
cells far exceeds that observed in the normal
population, an analytical method which enumerates such
cells should be sufficient. The determination of
patient status according to the methods described herein
is made based on a statistical average of the number of
circulating rare cells present in the normal population.
Levels of circulating epithelial cells in the early
stage cancer patient and in patients with aggressive
metastatic cancer can also be statistically determined
as set forth herein.
The following methods are provided to facilitate
the practice of the present invention.
Patients. With informed consent, 8-20 ml blood samples
were obtained from controls and patients with carcinoma
of the breast, prostate and colon. Blood was drawn from
some of these patients at several time points over a
period of one year. The blood samples were drawn into
Vacutainer tubes (Becton-Dickinson) containing EDTA as
anticoagulant. The samples were kept at room
temperature and processed within 24 hours after
collection. The circulating epithelial cells were
enumerated in peripheral blood samples from breast,
prostate and colon cancer patients and in normal
controls with no evidence of malignant disease. Date of
diagnosis, therapeutic interventions and clinical status
were retrieved from the patient's charts. The
institutional review board of the collaborating
institutions approved the protocol.
Sample preparation. Monoclonal antibodies specific for
36
~.,,..,...,,,..w..~......_......._._ _ , .._ - ---_._.._._
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
epithelial cell adhesion molecule (EpCAM) are broadly
reactive with tissue of epithelial cell origin (Stahel
RA, et al. Int J Cancer Suppl. 8:6-26 (1994); Momburg
F, et al. Cancer research. 47:2883-2891 (1987); Gaffey
MJ, et al. Am J Surg Path. 16:593-599 (1992)). The
GA73.3 or MJ37 EpCAM antibodies recognizing two
different epitopes on EpCAM (kindly provided by D Herlyn
(Herlyn D, et al. J Immunol Methods. 73:157-167
(1984)) Wistar Institute, Philadelphia, PA and MJ Mattes
(De Leij L, et al. Int J Cancer Suppl. 8:60-63 (1993))
Center for Molecular Medicine and Immunology, NJ) were
coupled to magnetic nanoparticles (ferrofluids) (Liberti
PA & Piccoli SP, United States Patent No. 5,512,332
(1996), Immunicon, Huntingdon Valley, PA). Blood was
incubated with the anti-EpCAM conjugated ferrofluid for
15 minutes in disposable tubes with an internal diameter
of 13 mm. The tubes were placed into a separator
composed of four opposing magnets for 10 minutes (QMS13,
Immunicon, Huntingdon Valley, PA). After separation,
the blood was aspirated and discarded. The tube was
taken out of the magnetic separator and the collected
fraction was resuspended from the walls of the vessel
with 2 ml of FACS permeabilization solution (BDIS, San
Jose, CA) and placed in the magnetic separator for 5
minutes. The solution was aspirated and discarded and
the cells were resuspended in 150 l of cell buffer
(PBS, 1% BSA, 50mM EDTA, 0.1% sodium azide) to which
phycoerythrin (PE) conjugated anti-cytokeratin (CAM5.2
Monoclonal antibody) and Peridinin Chlorophyll Protein
(PerCP)-labeled CD45 were added at saturating
conditions. After incubation for 15 minutes, 2 ml of
cell buffer was added and the cell suspension was
37
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
magnetically separated for 5 minutes. After discarding
the nonseparated suspension, the collected cells were
resuspended in 0.5 ml of the buffer to which the nucleic
acid dye used in the Procount system from BDIS, San
Jose, CA, was added according to manufacturer's
instructions. In some cases in which the EpCAM antibody
MJ37 was used on the ferrofluid, GA73.3 PE was used to
identify the selected epithelial cells. In these cases
no permeabilization of the cells is required. Reagents
for flowcytometry were kindly provided by BDIS, San
Jose, CA.
An exemplary method for determining 'the tissue
source of circulating epithelial cells employs
cytochemical and immunological identification
techniques. Primary monoclonal antibodies recognizing
cytokeratins 5, 6, 8, 18 (CK, 5D3, LP34, Novocastra),
MUC-1 glycoprotein (MUC-1, Ma695 Novocastra) or prostate
specific antigen (PSMA), clone J591 obtained from Dr.
Neil Bander (University of Texas Medical Center, Dallas,
Texas) was added to the slides after blocking non-
specific binding sites with 5% BSA for 30 minutes. The
samples were incubated for 20 minutes at room
temperature, washed twice in PBS for 5 minutes and then
exposed to secndary rabbit anti-mouse Ig (Z0259, Dako
Corp., Carpenteria, CA) for another 20 minutes. After
two more washes, the samples were incubated with
alkaline-phosphatase-anit-alkaline phosphatase (APAAP)
rabbit Ig complexes for 15 minutes. Finally, the
enzyme-substrate (New Fuchsin, Dako Corp. CA) was added
resulting in the development of red precipitates. The
nucleus was counterstained with hemotoxylin. The data
were recorded using a Kodak' digital camera attached to a
38
~........-..___ .... . _ .._.._..._
,,.~,...,.,.,_._,,,,~.,,._,,..____.. _._ _ ~_............m...._.. .._._.,._..~
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
light microscope. Data could be stored on CD for later
reference.
samvle analysia. 85% of the samples were analyzed on a
FACSCalibur' flowcytometer (BDIS, San Jose, CA). The data
were acquired in listmode using a threshold on the
fluorescence of the nucleic acid dye. Multiparameter
data analysis was performed using Paint-A-GateProT"` (BDIS,
San Jose, CA). Analysis criteria included size defined
by forward light scatter, granularity defined by
orthogonal light scatter, positive staining with the PE
labeled cytokeratin monoclonal antibody and no staining
with the PerCP labeled CD45 monoclonal antibody. For
each sample, the number of events present in the region
typical for epithelial cells was normalized to 10 ml of
blood.
The following examples are provided to facilitate
the practice of the present invention. These examples
are not intended to limit the scope of the invention in
any way.
EXAMPLE 1
Formulation of improved magnetic nanoparticles for the
efficient isolation of rare cells from whole blood
Rare cells (e.g., tumor cells in patients with
epithelial derived tumors, fetal cells in maternal blood
or the like) can be present in frequencies below one
rare cell per ml of blood. The number of blood smears
required to detect such rare cells is prohibitively
large. Assuming 10 rare cells in 10 ml of blood, which
39
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
corresponds to 10 tumor cells in 5-10 x 10' white blood
cells (leukocytes), cells can be transferred to a
microscope slide by cytocentrifugation or by settling,
stained with an antibody specific for the rare cells of
interest and read manually or automatically. The
maximum number of cells that can be transferred to one
slide is about 500,000 cells which means 100-200 slides
are required to process 10 ml of blood. The time
required for analysis by this approach makes it
impractical and economically unfeasible. Consequently,
enrichment methods such as sample volume reduction and
removal of erythrocytes and platelets by density
gradient separation or erythrocyte lysis procedures are
used for isolating rare cells so as to significantly
reduce the number of slides to be analyzed.
As noted above, magnetic enrichment is the
preferred method for cell separations and, ideally, the
nanoparticles employed for this purpose should not have
to be removed prior to analysis. Accordingly, the
nanoparticles should be small enough so as not to
interfere with analytical measurements, i.e. below about
250 nm. Most preferably, the nanoparticles are below 220
nm so as to make them filter sterilizable. Furthermore,
the nanoparticle should be large enough and magnetically
responsive enough to permit cell separation from simple
laboratory tubes, i.e., test tubes, centrifuge tubes,
vacutainers and the like in external gradient magnetic
separators. Again, as previously noted internal
gradient devices are cumbersome, costly and inefficient
for the recovery of rare cells. Also, the nanoparticles
and magnetic device should give high and reproducible
recovery with low non-specific binding. US Patent No.
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
5,597,531 describes the synthesis of highly magnetic
particles, referred to as direct coated (DC) particles
which have many of these characteristics. These
nanoparticles are composed of quasispherical
agglomerates of crystalline magnetite or other magnetic
oxides which are coated with polymers or proteins (based
coated magnetic particles). Because of their structure
(magnetic core and polymer coat where the core diameter
is >>> than the thickness of the coat) they are about
80-85% magnetic mass. The non-specific bindings of
these nanoparticles are in the range of 5-8 % and they
are, therefore, not very practical for rare cell
separations. Thus if one is enriching cells present at
one cell per ml then at 80% capture efficiency, the best
result to be expected using 10 mis of whole blood
(considering leukocytes alone) would be 8 cells
recovered in a total of 4 million, i.e. a 16-17 fold
enrichment. The magnetic particles described in U.S.
Patent 5,597,531 do, however, have the appropriate
magnetic properties to perform separations with open
field separators and from simple laboratory tubes.
Further, their mean size is well under the limit
suggested above and, hence, they do not interfere with
various analytical procedures. Based on extensive
studies with those materials, the major contributing
factor to non-specific binding to cells was discovered
to be the presence of bare crystalline iron oxides on
the nanoparticles due to incomplete coating. Such
incompletely coated crystals have a sufficiently high
positive charge at physiological pH that they are very
likely to bind strongly to biological macromolecules,
such as negatively charged sialic acid on cell surfaces.
41
CA 02432363 2003-07-03
WO 99/41613 PCT/US99103073
An improved method for making particles is described in
U.S. Patent No. 5,698,271. These materials are an
improvement over those disclosed in the 1531 patent in
that the process includes a high temperature coating
step which markedly increases the level of coating.
Nanoparticles made with bovine serum albumin (BSA)
coating using this process, for example, have a 3-5-fold
lower non-specific binding characteristic for cells when
compared to the DC-BSA materials of US Patent 5,579,531.
This decrease in non-specific binding has been shown to
be directly due to the increased level of BSA coating
material. When such nanoparticles were treated so as to
remove BSA coating, non-specific binding returns to high
levels. It was thus determined that a direct
relationship exists between the amount of BSA coated on
iron oxide crystal surfaces and the nonspecific binding
of cells. Typically, the non-specific binding of cells
from whole blood with these particles was 0.3% which is
significantly better than those produced from US Patent
5,579,531. Thus, from 10 mis of whole blood there would
be about 200,000 non-target cells that would also be
isolated with the cells targeted for enrichment.
In addition to the non-specific binding problem, to
be addressed further below, it was found that when
different lots of magnetic particles, manufactured as
described in US Patents Nos. 5,579,531 and 5,698,271
were used in rare cell depletions or enrichments,
recoveries were inconsistent. Sometimes recoveries were
85-95% and other times they could be 40-50% using the
same model system. As the process for manufacturing
these materials results in a size dispersion of
considerable range (30nm to 220 nm), it was suspected
42
CA 02432363 2003-07-03
WO 99/41613 PC'f/[1S99/03073
and confirmed that the size distribution and
particularly the presence of small nanoparticles
markedly affected target recovery. Since small
nanoparticles (30 to 70nm) will diffuse more readily
they will preferentially label cells compared with their
larger counterparts. When very high gradients are used,
such as in internal gradient columns, the performance of
these materials regardless of size make little
difference. On the other hand, when using external
gradients, or gradients of lesser magnitude than can be
generated on microbead or steel wool columns, the
occupancy of small nanoparticles on cells has a
significant effect. This was conclusively shown to be
the case by fractionating DC nanoparticles and studying
the effects on recovery. Based on these studies and
other optimization experiments, means for fractionating
nanoparticles magnetically or on columns was established
where base coated magnetic particles could be prepared
that were devoid of excessively small or large
nanoparticles. For example, base coated particles of
mean diameter 100nm can be produced which contain at
best trace amounts of material under 80 nm or over 130
nm. Similarly material of about 120 nm can be made with
no appreciable material under 90-95 nm and over 160 nm.
Such materials performed optimally with regard to
recovery and could be made sub-optimal by the inclusion
of 60-70 mm nanoparticles. The preferred particle size
range for use in practicing this invention is 90-150 nm
for base coated magnetic particles, e.g., BSA-coated
magnetite. Particles falling within this preferred
range may be obtained using the procedure described by
Liberti et al. In Fine Particles Science and Technology,
43
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
777-90, E. Pelizzetti (ed.) (1996).
To further address the non-specific binding
problem, several routes for making antibody conjugated
direct nanoparticles were attempted. Monoclonal
antibody specific for rare cells can be directly coupled
to, for example, the BSA base coating on the DC magnetic
particles by standard heterobifunctional chemistry
(referred to herein as direct coupling method).
Heterobiofunctional linkers used for these purposes
include sulfo-MCCC and sulfosuccinimidil-4-
jmaleimidomethyl]cyclohexane-l-carboxylate. In another
approach, biotinylated monoclonal antibodies can be
coupled to streptavidin which has been coupled to the
base coated particles. This conjugate method is
referred to herein as a piggyback method. In this
process, streptavidin is coupled to the base coated
magnetic particles by the same chemistry as the direct
coupling method. In one piggyback coupling method,
monobiotinylated antibody is allowed to react with
streptavidin magnetic particles for 1 hour and then the
remaining streptavidin binding sites quenched with free
biotin. It is important to quench the remaining
streptavidin sites after antibody coupling to prevent
binding of any biotinylated antibody to magnetic
particles during isolation of rare cells or the cell
analysis step. Furthermore, it has been shown that this
means for quenching streptavidin is effective for
counteracting non-specific binding. Incubation of such
materials under a variety of conditions with
biotinylated fluorescent macromolecules results in no
bound fluorescence. For comparison, anti-EpCAM antibody
(GA73.3 obtained from the Wistar Institute,
44
CA 02432363 2003-07-03
WO 99/41613 PCT/US99103073
Philadelphia, Pa.) was coupled to magnetic particles by
both methods. Both magnetic particles were then
compared for the selection of cells from the colon tumor
cell line (Colo-205) spiked into whole blood as well as
for the non-specific binding (NSB) or carry-over of
leukocytes. The leukocytes present in the final sample
were a combination of leukocytes non-specifically bound
to magnetic particles and carry-over of cells from the
wash steps. Note that following magnetic separation, it
is necessary to wash away any cells which were in
contact with the tube at the start of the separation or
that were transported non-magnetically during the
separation process. The following table shows the
comparison of those two magnetic particles.
Magnetic Recovery of NSB and carry over
particles spiked Colo-205 leukocytes (%)
cells M
EpCAM antibody
directly 78-82 0.1 - 0.3
coupled to
magnetic
particles
(lot.# 120325-
1)
EpCAM antibody
coupled to 67 - 78 0.05 - 0.1
magnetic
particles by
piggyback
method
(lot.# 120607-
2)
The first thing noted is that merely coupling
antibody or Streptavidin to BSA base particles
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
significantly reduces non-specific binding (data not
shown). This is believed to be due to decreasing the
accessability of "bare" crystal surfaces to cells for
binding. The above table demonstrates that the recovery
of spiked cells is comparable for both types of magnetic
particles. However, the non-specific binding of
leukocytes was 3-fold higher when using the direct
antibody coupled magnetic particles. This difference,
albeit relatively small, becomes significant when a
large volume of blood is processed and analyzed. A
reasonable explanation based on many supporting
observations for the difference between the two types of
magnetic particles is that there are more layers of
protein on magnetic particles synthesized using the
piggyback coupling method. The surface of the magnetic
crystals are thus coated more extensively with multiple
layers of protein and appear to be sterically
"protected". This prevents binding of non-target cells
to the magnetic particles.
In the piggyback coupling method, a limited number
of streptavidin binding sites on the magnetic particles
are occupied with biotin-antibody and the remainder are
saturated with free biotin by the quench process
described above. In yet another coupling method, the
excess streptavidin binding sites were quenched and
saturated with monobiotin-BSA instead of free biotin.
The rationale for this approach is that quenching with
monobiotin BSA should further sterically inhibit cells
from coming in contact with uncoated regions of the
nanoparticles, i.e. give better coverage of the
nanoparticles. It was shown by carbon analysis that
this process increases the amount of protein coupled to
46
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
the particles. The two magnetic particle preparations
were compared in experiments assessing recovery of
spiked Colo 205 from whole blood and for non-specific
binding of leukocytes. The results are presented in the
following table.
Magnetic particles Recovery of NSB and carry
Colo 205 over leukocytes
cells M ($)
EpCAM antibody 93 0.08
coupled magnetic 87 0.1
particles - 85 0.1
quenched excess
streptavidin sites
with free biotin
(lot. ## 131022-1)
EpCAM antibody 87 0.01
coupled magnetic 83 0.03
particles - 85 0.02
quenched excess
streptavidin sites
with biotin-BSA
(lot. # 131022-2)
Monobiotin-BSA may be prepared by conjugating a
limited amount of biotin to BSA, such that 30- 40% of
the resultant product has no bound biotin.
In summary, magnetic particles having a homogeneous
size distribution and biotin-BSA quenched streptavidin
binding sites performed extremely well in the assay
methods of the present invention. A good recovery of
the spiked epithelial tumor cells and almost an order of
magnitude reduction in nonspecific binding is obtained
using these particles, compared with the biotin blocked
nanoparticles. Thus, these materials and the results
obtained with them define a very useful product which
47
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
can be further optimized. The improved ferrofluid
product is made as magnetic as possible, is coated so as
to exclude all possible interactions of the magnetic
core with any substances in blood including cells
(presumably coated with a nonporous monolayer) and are
well defined in its size range and distribution. In the
preferred situation, a coat material is used which does
not interact with biological materials. Where such
interactions are unavoidable, a means for blocking them
is required. For a material to be as magnetic as
possible, those produced as described in US Patent Nos.
5,579,531 and 5,698,271 are preferred starting
materials. They are preferable because they are
composed of large magnetic cores with an apparent but
not complete monolayer of base coating material. For a
100 nm nanoparticle coated with BSA, the core will be
about 90 nm of an appropriate magnetic oxide such as
magnetite. Such nanoparticles because of the relative
size of the cores and coat material are clearly as
magnetic as is possible. This is apparent if one
considers that the function of the coating is to keep
the nanoparticles from undesired interactions with each
other which would lead to macroscopic agglomeration.
The coating also promotes sufficient interactions with
solvent molecules so as to maintain colloidal behavior
and provides a convenient chemical means for coupling.
The nanoparticles of US Patent Nos. 5,579,531 and
5,698,271 are also preferred as a starting material as
they have sufficient monolayer coating wherein "holes"
in the monolayer can be filled in several ways, viz.,
sterically and physically. Clearly any coating that
promotes the effective complete coverage of the magnetic
48
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
core, so as to inhibit interactions of the core material
with blood components or any other non-specific effects
in any other system would be suitable. The less mass
such a coating might add to the nanoparticles the
better, so as to maximize the magnetic mass to
nanoparticle mass ratio.
EXAMPLE 2
Enumeration of circulating epithelial cells in patients
treated for metastatic Breast Cancer
Figure 1 shows the results obtained when tumor
cells spiked into whole blood are isolated using the
assay methods of the present invention. Panel A shows
analysis by microscopy and panel B shows analysis
results obtained using flow cytometry. Fig. 2 shows
three examples of the flowcytometric analysis of 10 ml
blood samples obtained from one patient with metastatic
breast carcinoma at three time points, and includes the
correlative display of the anti-leukocyte versus anti-
epithelial cell antibodies of the flowcytometric
analysis. In Fig. 2, Panel A, 14 events are detected and
are present in the location typical for epithelial
cells. In Panel B, 108 epithelial cells are detected
and in Panel C 1036 epithelial cells are detected.
The number of events passing the threshold set
on the nucleic acid dye in the analysis of the 10-m1
blood sample varied between 5,000 and 50,000 events.
These events consist of cellular debris and leukocytes.
In analyzing the blood of 32 controls, the number of
events present in the region typical for epithelial
cells ranged from 0 - 4 / 10 ml of blood (mean = 1.0, SD
= 1.2).
49
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
Eight breast cancer patients had active metastatic
disease during the period of study. In these patients,
the number of epithelial cells in 10 ml of blood varied
within the range of 0 to 1036. The activity of the
disease was assessed by subjective criteria, i.e. bone
pain, dyspnea etc. and objective criteria, X-rays, bone
scans, CT scan, MRI and lymph node size. Patients were
classified in categories 0 through 4,as set out in Table
1.
TABLE I
Classification of patients according to clinical activity of the
disease after surgical intervention
Category Criteria
0 No evidence of disease at any time
point after surgical intervention
1 Evidence of disease at one time point
after surgical intervention
2 Evidence of disease under control
3 Active progressive disease
4 Life threatening disease
The dynamics of epithelial cell counts in -the
blood of 8 patients with metastatic disease are
presented in Fig. 3. The shaded area in the plots
indicates the range at which positive events were
detected in the controls. The plots also indicate when
chemotherapy was administered. Figure 3, panel A shows
a patient with life threatening disease and 200
epithelial cells / 10 ml of blood at the time` she
entered the study. High dose adriamycine reduced the
number within the normal range, but it rose again after
adriamycine was discontinued. After a second course of
adriamycine, the number of epithelial cells dropped
significantly, but was still above the normal range.
Fig. 3, Panel B shows the course of one patient over a
period of 43 weeks. The patient was asymptomatic at the
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
start of the study but was known to have bone metastasis
in the past. Epithelial cells were detected above
normal levels and steadily increased during the period
studied. A brief decline in the number of epithelial
cells was found after a course of high dose adriamycine
was administered. The activity of disease in this
patient clearly increased during this period. In Fig.
3, Panels C and D, two patients are shown with less
disease activity. In these patients, the changes in the
number of epithelial cells over time also reflected the
changes in the activity of the disease. In the patients
shown in Panels E and F, the number of peripheral blood
epithelial cells increased at the last time point
studied while the patients still were without symptoms.
In the case shown in Panel G, no epithelial
cells were detected at the first time point studied
which was three years after breast cancer surgery
(T2N1M0). Four weeks later, 50 epithelial cells in 10 ml
of blood were detected by flowcytometry. The patient at
this time had no clinical signs of disease recurrence.
An additional blood sample was analyzed to obtain
morphological confirmation that the cells detected by
flowcytometry had features consistent with those of
malignant cells.
Figure 4A shows two cells with a large nuclear
to cytoplasmic ratio and which positively stain with
Cytokeratin, both features being consistent with tumor
cells of epithelial cell origin. Four weeks after this
finding, the patient had an axillary lymph node biopsy.
Cells obtained from the biopsy proved to be of malignant
origin. Although an X-ray at this time did not show
signs of pulmonary metastasis, a CT scan performed two
51
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
weeks later showed evidence of pulmonary metastasis.
The patient had no symptoms from the pulmonary
metastasis. The patient reacted well to Vinorelbine as
measured by the disappearance of the axillary lymphnode
involvement. The peripheral blood epithelial cell
number dropped to levels just above the normal range.
Twenty-eight weeks after initiation of the treatment,
the peripheral blood epithelial cell number increased
and by physical examination, the axillary node increased
in size. The number of peripheral blood epithelial
cells in these 8 patients with metastatic disease of
carcinoma of the breast clearly reflected the activity
of the disease and the response to treatment or the lack
thereof during the time period studied.
The experiments described above were performed
using colloidal magnetic nanoparticles. In this
example, the efficiency of larger size magnetic beads
for the selection of tumor cells present at a low
frequency in blood was also evaluated to determine
whether micron size beads can also be used to select
tumor cells even though as described above, nanometer
size magnetic particles are considered preferable for
this application.
As mentioned previously, disadvantages are
encountered with the use of larger size beads. These
are: (i) the beads are too large to diffuse thus
collisions of the beads with target cells present at a
low frequency requires mixing (ii) the beads settle
very fast, furthering the need for continuous mixing and
(iii) large size beads cluster around cells and obscure
analysis. Accordingly the large size beads need to be
removed from the cell surface prior to visualization or
52
,_,,,_,_,.,.,,~....,._._~,._..,,,,.,.,.....,.._.....+.,......___ . _ _ . _
__.._..__.._. ~_.._ .
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
analysis. In accordance with the present invention, it
has been found that the efficiency of cell selection
with larger beads can be improved by increasing the
concentration of beads and increasing the incubation
time with continuous mixing to facilitate binding to
rare target cells. In this example, 2.8 m Dynal
anti-epithelial cell beads (Dynal, NY) were used to test
the efficiency of tumor cell selection from blood in a
model study under optimum conditions for large beads.
These beads are conjugated with a monoclonal antibody
specific for epithelial tumor cells. A known number of
tumor cells (cancer cell line) were spiked into normal
blood to determine the recovery after selection with
beads. The tumor cells were prelabeled with a
fluorescent dye to differentiate them from blood cells
during detection. The protocol was followed as
recommended by the manufacturer.
Whole blood (5 ml) was added to a 15 ml
polystyrene centrifuge tube followed by the addition of
20 3 fluorescently labeled SKBR-3 (breast cancer cell
line) cells. SKBR-3 cells were prestained with a nucleic
acid staining dye (Hoescht) to allow detection after the
selection by beads. The blood was diluted with 5m1 of
Dulbecco's PBS containing 5 mM EDTA and mixed with the
diluted blood for 15 minutes at 4 C on a rocker. 100 1
of Dynal anti-epithelial cell beads containing 50 x 106
beads were added to the blood sample and iricubated for
minutes at 4 C with mixing on a rocker. Note that the
number of beads used were similar to total white blood
30 cells i.e. one bead per white cell. The magnetically
labeled cells were separated by placing the sample tube
into Dynal MPC magnetic separator for 6 minutes.
53
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
After aspirating the supernatant, the
collected cells were resuspended in 3ml of Dulbecco's
PBS containing 0.1% BSA. The sample tube was placed back
into Dynal's MPC for 6 minutes to remove any carry-over
blood cells. The magnetically bound cells were
resuspended in 200 l of Dulbecco's PBS containing 0.1%
BSA after aspiration of the supernatant.
The final sample, containing selected tumor
cells, non-specifically bound blood cells and excess
free magnetic beads, was spotted onto an
immunofluorescent slide to detect tumor cells. The 200 1
sample was spotted into 10 different wells to disperse
free magnetic beads. The fluorescently stained tumor
cells present in each well were counted using a
fluorescent microscope. The results are shown in the
Table II:
TABLE II
Experiment No. Tumor cells recovered % Recovery
1 16 80
2 17 85
3 10 50
4 11 54
The results show that, on average, 67% of the
spiked tumor cells were recovered from blood by Dynal
magnetic beads. This suggests that tumor cells present
in blood can be selected efficiently with larger size
magnetic beads under optimum conditions. I:n this
example, however, only the selection of tumor cells from
blood was evaluated without performing any analysis.
Further the efficiency of recovery could be determined
because cells were prelabeled with a strong fluorescent
54
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
dye. The final sample (200 1) contained 50 x106 beads in
addition to selected tumor cells (10-17) and
non-specifically bound leukocytes. The size of the beads
(2.8um) is similar to that of certain blood cells and
occupied most of the surface area on the slide.
Therefore, to obtain recovery data, the sample had to be
spotted onto several wells in order to sufficiently
disperse free magnetic beads so as to allow for
detection of recovered tumor cells.
There were also many beads on cell surfaces
which precludes viewing and staining of selected tumor
cells for further analysis. In this example, tumor cells
were prestained with a fluorescent nucleic acid dye and
further staining was not necessary for detection.
However it is often desirable to identify the tissue of
origin of the magnetic bead-bound cells. Such
identification is performed using labeled antibodies to
detect and characterize tumor cells present in clinical
samples. Accordingly, beads have to be removed from
cell surfaces and separated from the sample following
target cell selection, i.e. before analysis. This is not
the case with nanosize magnetic particles because their
size does not interfere with cell analysis.
In summary, this example shows that large
magnetic beads may also be utilized in the methods
disclosed herein for the efficient isolation of
circulating tumor cells.
There are several methods available to release
beads from cell surfaces which do not significantly
damage isolated cells. One method is to displace
antibody from the cell surface by adding an excess
specific competing reagent in excess which has higher
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
affinity for the involved antigen or antibody. This
type of mechanism is used to release beads from CD34
selected cells in clinical applications using a peptide
(Baxter Isolex 300). The peptide competes with CD34
antigen for binding to antibody on beads and releases
the antibody-bead complex from cells. Another method
employs a reversible chemical linker between beads and
antibodies.
The chemical linker can be inserted during
the conjugation of antibodies to magnetic beads. The
chemical link can be cleaved under appropriate
conditions to release beads from antibodies. One of
the methods currently in use employs a nucleic acid
linker to link antibodies to magnetic beads. The
nucleic acid linker is a polynucleotide and can be
hydrolyzed specifically using DNAse enzyme. Following
hydrolysis of the nucleotide bonds present in the
nucleic acid linker, the beads are released from the
antibodies which remain bound to cells. The released
beads can be removed from cell suspension by magnetic
separation. The cells which are freed from beads can
be used for further analysis by microscopy or flow
cytometry.
This example demonstrates that larger size
magnetic beads can also be used to isolate tumor cells
from blood, provided they are used in high enough
concentration to label cells and are then released
from cells before analysis.
56
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
EICAMPLE 3
Enumeration of circulating epithelial cells in patients
with no evidence of disease after surgery for carcinoma
of the breast with curative intent
Peripheral blood of 37 patients between 1 and 20
years after surgery was examined for the presence of
epithelial cells by flowcytometry. Up to 7 peripheral
blood samples were taken over a one-year period from
these patients. In Table III, each of the patients is
listed and sorted according to the TNM (tumor, node, and
metastasis) stage at the time of surgery followed by the
years after surgery. Table III also shows whether or
not the patient received treatment (either chemotherapy
or hormonal therapy) during the period studied. In 3 of
6 patients with evidence of distant metastasis in the
past, but in complete remission at the time of study,
epithelial cells were found in the blood at a higher
frequency than that found in the control group.
Circulating epithelial cells were also found in 9 of 31
patients with no evidence of distant metastasis.
The low number of events present in the region
typical for epithelial cells by flowcytometry in these 9
patients does not warrant identifying these events as
tumor cells. Cytology obtained by placing the
immunomagnetically selected cells on a slide greatly
aids in the assessment of their identity as is
illustrated in Fig. 4. Fig. 4, panel A, shows two cells
staining positive for cytokeratin and obtained from a
patient with no evidence of metastatic disease at the
time the blood was drawn. Panel B shows a cell from a
patient with metastatic disease in the past but in
complete remission. In Panels C and D, two cells are
57
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
shown isolated from the blood of patient 25 at time
point 6. The cell shown in Panel C has features
consistent with malignancy whereas the cell in Panel D
has the appearance of a normal squamous epithelial cell.
58
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
TABLE III
Number of epithelial cells identified by flowcytometry
in 10 ml of peripheral blood of patients with no
evidence of disease after surgery for carcinoma of the
breast with curative intent and 32 controls.
Patient
Number TNM Ys Tx 1 2 3 4 5 6 7
1 T3N1M1 9 - 2 29
2 T3N1Mi 16 H 0
3 TZN1M1 7 CT 10 7 5 6 4 8 7
4 TZN1M1 10 CT 1 0 0 1 2
5 TZN1M1 10 H 6
6 TZN1M1 20 H 12 2
7 T3N1Mo 1 H 0
8 T3N1Mo 2 CT 0 0 0
9 T3N1Mo 2 CT 0 0 1 0
10 T3N1Mo 3 H 3
11 T3N1Mo 3 H 5 4 0 6
12 T3N1Mo 3 H 3 0
13 T3N1Mo 6 CT 6 0
14 T3N1Mo 6 H 1 1
15 T3N1Mo 7 H 1 3 3
16 T3N1Ma 3 H 0
17 TZN1Mo 17 H 4
18 T3NoMo 3 - 5
19 T3NoMo 5 H 1
20 T3NoMo 8 H 0 6 8
21 TZMoMo < 1 - 0
22 TZNoMo <1 H 0
23 TzNaMo 1 H 0
24 T2NaMQ 1 - 4
25 TZNoMo 2 CT 3 5 1 3 6 2
26 TZNoMo 3 CT 2 6 3 1 1 0 5
27 T2NoMo 6 H 18
28 TzNoMo 6 H 2 1
29 TZNoMo 7 H 8 4 2
30 TZNoMo 8 H 0 1
31 T2NoMo 8 H 0 6 8
32 T2NoMa 11 H 2
33 T2NoMo 20 H 4
34 T1NoMo <1 H 0
35 T1NoMo 2 H 0
36 T1NoMo 17 - 0
37 T,NoMa 13 H 0
N3 controls
2 min 0
59
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
max 4
mean 1.0 M 2SD 3.5
TNM = Tumor, Node, Metastasis
Ys = years after primary surgery
Tx = therapy, CT = chemotherapy, H = hormonal therapy, -
= no therapy
1,2,3,4,5,6,7 = subsequent time point at which the
number of epithelial cells was determined in years
EXAMPLE 4
Enumeration of circulating epithelial cells in patients
diagnosed with breast cancer before surgical
intervention.
Table IV summarizes the results obtained
following similar clinical trials in which 13 controls
and 30 patients with breast cancer were assessed using
the assay of the invention. In control individuals the
number of epithelial cells in 20 ml of blood ranged from
0-5 (mean 1.5 f 1.8). In contrast, there was an average
of 15.9 f 17.4 epithelial cells in the 20 ml blood
samples of 14 patients with organ-confined carcinoma of
the breast (patients classified as T.NoMo) , 47.4 f 52.3
in those with nodal involvement, and 122 140 in those
with distant metastases. The difference between the
control group and patients with carcinoma of the breast,
with or without metastasis, was highly significant
[P,0.001 by multiparameter analysis (Kruskal.-Wallis)).
The difference between the organ-confined and the
distant metastatic group was 0.009(t test). The number
of epithelial cells in patients with organ-confined
breast cancer was above the cut-off point (mean value
plus 3 SD in the control group = 6.9) in 12 of 14 cases.
Moreover, no individual in the control group had more
than 5 events classified as epithelial cells, and only 2
_~..~
__ ___ _ -......_.._._._._..._....r-.._.~...._..._ ..._.._...,._ ____ ._._
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
of the 14 patients with organ-confined breast cancer had
<7 such events.
Table IV
Sunrmary of clinical data
8ealthy No detectable Spread to Distant
Number Control spread lymphnodes metastasis
only
1 0 0 7 20
2 0 4 8 20
3 0 7 14 20
4 0 8 93 23
5 0 8 115 54
6 0 8 62
7 0 12 99
8 2 13 135
9 2 14 152
10 4 16 304
11 4 18 456
12 5 19
13 24
14 72
n 12 14 5 11
mean 1.5 15.9 47.7 122.5
Flowcytometry was used to analyze the positive events
obtained from 20 ml of blood from control individuals
and from women with breast carcinoma. The numbers of
epithelial cells in the blood of controls are
statistically different by t test (P s 0.01) and by
Kruskall-Wallis nonparametric analysis (P<0.001) from
each of the three groups of the breast cancer patients.
The data in this table were used to establish a
preliminary cut-off value for positive samples. This
61
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
value was determined by averaging the number of
circulating epithelial cells in the normal controls (n
=13) and then adding three times the SD. The average
was 1.5 and the SD is 1.8. Cut-off: 1.5 + 5.4 = 6.9.
There is no statistical difference between male and
female controls.
EXAMPLE 5
DISEASE ACTIVITY IS CORRELATABLE WITH NUMBER OF
CIRCULATING EPITHELIAL CELLS IN PROSTATE CANCER PATIENTS
Three patients with metastatic disease of the
prostate were assessed for the presence of circulating
epithelial cells in their blood following
chemotherapeutic treatment. The results are presented
in Fig. S. The data reveal that an increase in
circulating epithelial cells in the blood is
correlatable with disease activity. Also in three
patients with no detectable spread of the cancer,
epithelial cells were found in 20 ml di the peripheral
blood (16 cells 4). As shown in Table V, the number of
epithelial cells in the blood of prostate cancer
patients was statistically different (P less than 0.001)
than normal controls.
TABLE V
Number of Epithelial PSA Gleason
Cells Per 20 ml Blood Level Grade
( g/ml )
4 6.0 3 + 4 =7
12 6.5 3+ 4=7
12 11.2 3 + 3 =6
16 26.0 4 + 4 =8
24 5.6 3 + 4 =7
35 28
62
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
Control blood samples were obtained from normal
individuals, individuals known to have benign tumors and
those patients with inflammatory diseases. Based on
statistical data, the results reveal that a cut-off
point of approximately 6.8 cells per 20 ml of blood was
useful as a diagnostic marker for prostate cancer.
EXAMPLE 6
DISEASE ACTIVITY IS CORRELATABLE WITH NUMBER OF
CIRCULATING EPITHELIAL CELLS IN COLON CANCER PATIENTS
The assay method of the present invention may be
used to advantage in the assessment of patients with a
variety of different cancer types. To illustrate, the
method was also used to assess circulating epithelial
levels in patients with colon cancer. Colon cancer
patients without evidence of metastases were evaluated
for the presence of circulating epithelial cells before
and after surgery. The results are shown in Fig. 6 and
summarized in Table VI. The data reveal that the number
of circulating epithelial cells in colon cancer patients
is greater prior to surgical intervention.
TABLE VI
CIRCULATING EPITHELIAL CELLS IN COLON CANCER PATIENTS WITHOUT
EVIDENCE OF METASTASES
TIME OF NUMBER OF CIRCULATING EPITHELIAL CELLS
TESTING PATIENTS DETECTED BY FLOW CYTOMETRY IN
TESTED 10 ml OF BLOOD
MEAN t SEM RANGE
Before surgery 12 42.3 f 22.0 0 - 234
After surgery 25 2.7 ~ 0.7 0 - 15
Table VII and Fig. 7 depict data obtained when
colon cancer patients with evidence of metastases were
63
õ~..._.~õ~...,~.,,.... -....~.~....._..__-____.._~ -.~_._ _
CA 02432363 2003-07-03
WO 99/41613 PCT/tJS99/03073
assessed for the presence and number of circulating
epithelial cells. The results revealed that the number
of epithelial cells in peripheral blood is larger in
patients with metastatic disease as compared to local
disease after surgery. The results further show that
the extent of metastatic disease may be correlated with
the number of circulating epithelial cells.
TABLE VII
CIRCULATING EPITHELIAL CELLS IN COLON CANCER PATIENTS WITH EVIDENCE
OF METASTASES
METASTATIC NUMBER OF CIRCULATING EPITHELIAL
STATUS OF PATIENTS CELLS DETECTED BY FLOW
PATIENTS TESTED CYTOMETRY IN 10 ML OF
TESTED BLOOD
MEAN t SD RANGE
REGIONAL 11 3.7 t 0.6 1 - 6
DISTANT, SOLITARY 16 7.6 f 2.0 0 - 21
DISTANT, MULTIPLE 8 54.0 f 25.1 5 - 200
NORMAL CONTROL 32 1.0 ,t 0.2 0 - 4
The examples above demonstrate the highly-
significant differences in the number of circulating
epithelial cells between healthy individuals and
patients with breast, prostate and colon cancer. In
addition, significant differences in the number of
circulating epithelial cells were found between patients
with no detectable spread, spread to local lymph nodes
and distant metastasis (Racila et al., (1998), supra).
Additionally, the number of epithelial cells in the
blood of patients after surgical removal of a primary
carcinoma of the breast was monitored over a one-year
period. In some of these patients residual. disease was
detected. See Figure 7. In patients with metastatic
disease, the changes in peripheral blood tumor cell
count correlated with the tumor load and response to
64
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
treatment. The results of these studies reveal the
potential of the cell-based assay of the present
invention as an objective non-invasive tool to detect
the presence of malignant disease and measure the
activity of the disease. Cellular morphology and
immunophenotype reveal the malignant nature of the
isolated cells.
EXAMPLE 7
TISSUE SOURCE IDENTIFICATION OF ISOLATED EPITHELIAL
CELLS
All of the forementioned studies in patients reveal
that there is an excess of epithelial cells in patients
who have cancer, compared to normal individuals or
patients without cancerous diseases, including benign
tumors. It is essential, however, to prove that these
excess epithelial cells are, in fact, cancer cells.
This was accomplished by performing an experiment in
which immunomagneticallly purified epithelial cells from
patients with or without cancer were cytospun onto a
glass slide and treated with anti-mucin. In addition,
normal epithelial cells which were obtained from
foreskin and blood from normal individuals, both used as
controls, were also cytospun. It is significant that
the slides were coded and examined "blinded", that the
observer had training in pathology and that normal
epithelial cells were included. As can be seen in
Figure 4, there is a marked difference between the
cancer cells versus normal epithelial cells. Normal
epithelial cells have a low nuclear to cytoplasmic
ratio, i.e., there is abundant cytoplasm and a
relatively small nucleus. The nucleus shows a smooth
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
distribution of chromatin. The cells do not stain with
anti-mucin. In contrast, cells from two patients with
breast cancer have very large nuclei and a. small rim of
cytoplasm. Additionally, the chromatin is disorganized
as shown by the dark patches in the nucleus and the
cells stain intensively with anti-mucin. The same is
observed in cells from two patients with prostate
cancer. A physician trained in pathology was shown
coded slides from patients with and without cancer
(total of 21 slides). The pathology-trained physician
correctly identified bloods from all the controls as not
having cancer cells and displayed no-intraobserved error
when shown slides twice. In the cases of two patients
with prostate cancer, tumor cells were not seen in the
study. One slide was re-examined and tumor cells were
observed. The cause of this discrepancy appears to be
the amount of time spent scanning the cell smear. In
summary, the cytomorphology and immunophenotype indicate
that the excess epithelial cells present iri the blood in
patients with cancer are indeed cancer cells.
The experiments described above indicated that
the methods disclosed herein enable the detection of
cancer cells in the blood of patients with early tumors.
Indeed, in 25 of 27 patients who were clinically
determined to have organ-confined disease (early stage
cancer), we detected the presence of cancer cells in the
blood. This means that the assay should detect cancer
cells much earlier in those solid tumors that are
normally detected late (109-1010 tumor cells) . Morever,
the test should allow detection of breast and prostate
cancer earlier, perhaps before detection of a primary
tumor by conventional means. The organ-origin of tumor
66
...~_...~_. - --..,....._.....,-_~--.
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
cells in the blood for prostate can be established by
staining with anti-prostate specific membrane antigen
(PMSA), anti-PSA (prostate specific antigen), or other
antibodies specific to the prostate in male subjects.
For breast carcinoma in female patients, st.aining with
anti-mammoglobin, anti-progesterone receptor, anti-
estrogen receptor and anti-milk fat globulin antigen I
and II will indicate a breast origin of tumor.
Our test should detect carcinoma cells from
other organs, e.g., pancreas, esophagus, colon, stomach,
lung, ovary, kidney, etc. The following table shows
examples in which excess epithelial cells were observed
in several patients with carcinomas other that the
breast and prostate.
TABLE VIII
NUMBER OF CELLS CANCER
PER 20 ML BLOOD DIAGNOSIS
8 Uterus adenocarcinoma
(Stage 1B)
11 Head and Neck
adenocarcinoma
15 Lung small
undifferentiated
14 Neck Squamous cell
carcinoma
Each of the carcinomas described in the table
above express tissue specific antigens whose
corresponding antibodies can be used to determine the
organ-origin of the circulating tumor cells.
The blood test of the invention can also be
used to detect cancer cells in patients previously
treated successfully for cancer and now in long term
complete remission. Indeed circulating epithelial
cells, i.e., dormant tumor cells, have been detected in
patients treated five or more years previously and who
appear to be clincally free of tumor. This explains why
67
...__.. ._,... _,_.w_.._..._.....~.....~....-
......o.....,.........+.......~..~.-e-,-
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
recurrence in patients can occur many years, even
decades after apparently successful treatment. In fact,
accumulating evidence suggests that the recurrence rate
of breast cancer is at slow steady rate 10-12 years
after mastectomy.
EXAMPLE 8
DETECTION OF TUMOR CELLS IN THE BLODD OF A PATIENT WITH
HIGH PSA LEVELS AND A NEGATIVE BIOPSY
As indicated by the foregoing examples, the
present invention may be used to advantage to diagnose
cancer in presently asymptomatic patients. To
illustrate this point, a patient with a two year history
of high PSA levels (>12 g/ml), had a needle biopsy of
the prostate performed two weeks prior to the analysis
set forth below. The biopsy did not reveal the presence
of malignancy. It is also noteworthy that a prior
biopsy performed 18 months earlier was also negative.
Before obtaining a 20 ml blood sample, the
patient was given a digital rectal exam and a gentle
massage of his enlarged prostate with the intention of
increasing the occurrence of zumor cells in the blood.
The blood sample was enriched using the methods of the
present invention. The enriched fraction was examined
by microscopy employing a Wrights-Giemsa stain.
Morphological examination of the isolated cells revealed
their malignant character. Clearly this patient had
cancer. Given the high PSA levels observed, a diagnosis
of prostate cancer is likely. The origin of the cells
may be determined using appropriate reagents as
described herein. The results presented in this example
68
-___--_,_õ~.~...~..._..,..,.._._.____.A.._......_ . ..
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
reveal that the methods of the present invention can be
used to detect cancers which might otherwise go
undetected.
The notion of employing a localized massage to
promote shedding of tumor cells into blood as a means of
enhancing sensitivity of the blood test is a concept
with considerable merit. Cells that are released into
the circulation by this approach, following isolation
may be used for a variety of different purposes. In the
case of cells isolated with ferrofluids, isolated cells
can be readily cultured and/or cloned. The resultant
cell lines can be used to assess a variety of malignant
cell characteristics such as chemotherapeutic
sensitivity and growth factor dependency.
EXAMPLE 9
Tests Kits for diagnosing various aspects of cancer.
Also contemplated for use in the present
invention are test kits comprising the reagents used to
perform the assay of the invention. Such kits are
designed for particular applications. Reagents may be
assembled to facilitate screening of patients for
circulating rare cells, including but not limited to
tumor cells. In this embodiment, the kits contain
colloidal magnetic particles comprising a magnetic core
material, a protein base coating material and a
biospecific ligand which binds specifically to a
characteristic determinant present on the cancer cell to
be isolated. The kit also includes at least one
additional biospecific reagent which has affinity for a
second characteristic determinant on the cancer cell to
be isolated which differs from the determinant
69
,.~,_...,,_...,..~...._._.____.._-..._ .__._~..._...~
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99103073
recognized by the biospecific ligand. The kit also
includes a cell specific dye for excluding non-nucleated
cells and other non-target sample components from
analysis.
A typical kit according to this invention may
include anti-EpCAM coupled directly or indirectly to
magnetic nanoparticles, and a pair of monoclonal
antibodies, the first antibody recognizing a cancer
specific determinant and the second antibody having
affinity for a non-tumor cell determinant, e.g., a pan
leukocyte antigen. The kit also contains a nucleic acid
dye to exclude non-nucleated cells from analysis. The
kit of the invention may optionally contain a biological
buffer, a permeabilization buffer, a protocol,
separation vessels, analysis chamber, positive cells or
appropriate beads and an information sheet.
The kits described above may also be produced
to facilitate diagnosis and characterization of
particular cancer cells detected in circulation. In
this embodiment, the kits contain all of the items
recited above, yet also preferably contain a panel of
cancer specific monoclonal antibodies. Using breast
cancer as an example, a kit for diagnosis may contain
anti-MUC-1, anti-estrogen, anti-progesterone receptor
antibodies, anti-CA27.29, anti-CA15.3, anti-cathepsin D,
anti-p53, anti-urokinase type plasminogen activator,
anti-epidermal growth factor, anti-epidermal growth
factor receptor, anti-BRCA1, anti-BRCA2, anti-prostate
specific antigen, anti-plasminogen activator inhibitor,
anti-Her2-neu antibodies or a subset of the above.
A kit is also provided for monitoring a
patient for recurring disease and/or residual cells
.,,,........_.Y.,._,.._.....,-..,,.._........ _...___ _...._...,. -..-,-..-
_.~.._ ........... ...
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
following eradication of the tumor. In this embodiment,
the type of cancer will already have been diagnosed.
Accordingly, the kit will contain all of the reagents
utilized for screening biological samples for cancer yet
also contain an additional antibody specific for the
type of cancer previously diagnosed in the patient.
Again using breast cancer as an example such a kit might
contain anti-MUC-1. Alternatively, the kit may contain
anti-Her2-neu.
The kits of the invention may be customized
for screening, diagnosing or monitoring a variety of
different cancer types. For example, if the kits were
to be utilized to detect prostate cancer, the antibodies
included in the kit would be specific for prostate
tissue. Suitable antibodies or markers for this purpose
include anti-prostate specific antigen, free PSA,
prostatic acid phosphatase, creatine kinase, thymosin b-
15, p53, HPC1 basic prostate gene and prostate specific
membrane antigen. If a patient were to be screened for
the presence of colon cancer, an antibody specific for
carcinoembryonic antigen (CEA) may be included in the
kit. Kits utilized for screening patients with bladder
cancer may contain antibodies to nuclear matrix protein
(NMP22), Bard Bladder tumor antigen (ETA) or fibrin
degradation products (FDP). Markers are known for many
different cancer types.
The cells isolated using the kits of the
invention may be further studied for morphology, RNA
associated with the organ of origin, surface and
intracellular proteins, especially those associated with
malignancy. Based on existing information on such
molecules, it should be possible to determine from their
71
...............,..,..._.......Y_.,._..._...-...-,..---...._----_... __
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
expression on the isolated cell, the metastatic
potential of the tumor via analysis of the circulating
cells.
It is an object of the invention to provide
kits for any cancer for which specific markers are
known. A list summarizing those markers known at this
time and the usefulness and/or indication f'ollows:
I Indicative of tumor origin
Muc-1 -- breast
PSA, PSMA -- prostate
CEA -- colon
CYPRA 21-1 -- lung
CA 125 -- ovarian
cytokeratins -- see list
anti-HI67
II Cell cycle
nucleic acid dye
cyclin A, C & E
p27
III Cell viability/apoptosis
Fas (CD95)
amexin V
anti-metalloproteinases
IV Drug sensitivity
estrogen, progesterone & androgen receptors
HER-2/neu
V. Drug resistance
P-glycoprotein (MDR)
t-glutamylcysteine synthase
taxol-resistance-associated-gene-1-5
cis-diamminedichloroplatinum II resistance genes
thymidylate synthetase
protein kinase C
telomerase
VI. Staging
Lewis A
C
BRCA-1 BRCA-2
CA15.3 (Muc-1), CA 27.29, CA 19.9
LASA
72
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
p53
cathepsin D
ras oncogene
The following table provideS different cytokeratin
markers that may be used to assess tissue origin of
cells isolated using the methods of the present
invention.
TABLE IX
CYTOKERATIN MARKERS
Cytokeratin Number lkl3kJ5161718 10 11 12 13 14 15 16 17 18 19 20
Adrenal Cortex - - - - - + - - - - - - - - - + + -
Endometrium - - - - - - + + - - - - - - - - - + + -
Esophagus - - - - - + - - - + - - - - + + -
Gastro-Intestinal - - - - - + - - - - - - - - - + + +
Kidney - - - + + - - - - - + + + -
Liver ----- ++ - - - - - - - + + -
Lung Columnar ------++- - - - - - - - - + + +
Lung Basal ---- -- - - - - - + + - + - - -
Mammary Gland Luminal --- --+ +- - - - - - - + + -
Mammary Gland Basal - - - - - - - - - - - + - +
Mesothelium ---- ++- - - - - - - - - + + -
Oral ------- -- - - - - - - + - - -
Ovary ------ ++ - - - - - - - + + -
Pancreas - - - - + + - - - - - + - - + + +
Pituitary Endocrine cells --- --- +- - - - - - - - - + - -
Pituitary Follicular cells - - - - - - + - - - - - - - - - - - + -
Prostate Basal - - - - - + - + - - + + - - + + + -
Prostate Luminal - - - - - - + - - - - - - - - + + -
Skin ------- -- - - - - - - - - - -
Testis -- --- - + - - - - - - - - - + - -
Thymus ---- - - + - - - - - - - + - +
Thyroid --- -- - +- + - - + - - - - + + -
Urinary Bladder - - - - + + - - - - + - - - + - +
Uterine Cervix - - - + + - - - - - + + + + + +
Non-Epithelial:
Mammary adenocarcinoma ------++ - + -
Prostate adenocarcinoma + + + -
Pancreatic adenocarcinoma + + + + +
Gastro-Intestinal
---- ++- - - - - - + + +
adenocarcinoma
73
...~...~~.._õ~....,._..--~...-...._ .._.......... _._
....._.._.__..._...___...._._.__-__..
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
Endometrium adenocarcinoma - - - - + + - - - - - + +
Lung adenocarcinoma --- --+ +- - - - 1- 1- + +
Lung SCC - - - + - - - + + + + + +
- - - - - + + -
Kidney renal cell tumor --+ + + Oral SCC + + - - - - - + -
Liver --1-1-1-1+
Ovary --+ - + + +
Pituita
ry adenoma --- - - - - - + Testis --10 Thyroid + + + + Urinary Bladder --+
uterine cervix + + + valvular carcinoma + - - + + - - - - -
The following demonstrates how the practice of the
methods of the invention is facilitated by means of a
kit for use in detection of circulating breast cancer
cells:
As described above, the kit starts with reagents,
devices and methodology for enriching tumor cells from
whole blood. The kit would contain reagents to test for
breast cancer cells in a blood sample which will assess
six factors or indicators. The analytical platform
needs to be configured such that the reporter molecules
DAPi, CY2, CY3, CY3.5, CYS, and CY5.5 will be
discriminated by the appropriate excitation and
emmission filters. The analytical platform in this
example uses a fluorescent microscope equipped with a
mercury arc lamp, and the appropriate filter sets for
assessing the wavelengths of the detection labels
employed. All of the markers are introduced at one time
with this method. DAPi, which is excited with Uv light,
stains nucleic acids, and will be used to determine the
nuclear morphology of the cell. CAM 5.2 labelled with
CY2 will be used to stain the control cells. CY3
74
- _.....__ __.-w.._......_ _ _......... _..-_._..,.
......_...~~....,~..~.....~-.,..._..
CA 02432363 2003-07-03
WO 99/41613 PCTIUS99/03073
labelled Cll will be used to label cytokeratins 7, 8,
18, and 19. An antibody conjugated with CY3.5 will be
used to label HER-2/neu. An antibody conjugated with
CY5 will be used to label Muc-1. An antibody conjugated
to CY5.5 will be used to label estrogen receptors. By
using the appropriate excitation and emmission filters,
the cancer cells will be identified.
Examples of different types of cancer that may be
detected using the compositions, methods and kits of the
present invention include apudoma, choristoma,
branchioma, malignant carcinoid syndrome, carcinoid
heart disease, carcinoma e.g., Walker, basal cell,
basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in
situ, Krebs 2, merkel cell, mucinous, non-small cell
lung, oat cell, papillary, scirrhous, bronchiolar,
bronchogenic, squamous cell and transitional cell
reticuloendotheliosis, melanoma, chondroblastoma,
chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant
cell tumors, histiocytoma, lipoma, liposarcoma,
mesothelioma, myxoma, myxosarcoma, osteoma,
osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma,
adenolymphoma, carcinosarcoma, chordoma, mesenchymoma,
mesonephroma, myosarcoma, ameloblastoma, cementoma,
odontoma, teratoma, throphoblastic tumor,
adenocarcinoma, adenoma, cholangioma, cholesteatoma,
cylindroma, cystadenocarcinoma, cystadenoma, granulosa
cell tumor, gynandroblastoma, hepatoma, hidradenoma,
islet cell tumor, leydig cell tumor, papilloma, sertoli
cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma,
myoblastoma, myoma, myosarcoma, rhabdomyoma,
rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma,
medulloblastoma, meningioma, neurilemmoma,
CA 02432363 2003-07-03
WO 99/41613 PCT/US99/03073
neuroblastoma, neuroepithelioma, neurofibroma, neuroma,
paraganglioma, paraganglioma nonchromaffin,
antiokeratoma, angioma sclerosing, angiomatosis,
glomangioma, hemangioendothelioma, hemangioma,
hemangiopericytoma, hemangiosarcoma, lymphangioma,
lymphangiomyoma, lymphangiosarcoma, pinealoma,
carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes,
fibrosarcoma, hemangiosarcoma, leiomyosarcoma,
leukosarcoma, liposarcoma, lymphangiosarcoma,
myosarcoma, myxosarcoma, ovarian carcinoma,
rhabdomyosarcoma, sarcoma (Kaposi's, and mast-cell),
neoplasms (e.g., bone, digestive system, colorectal,
liver, pancreatic, pituitary, testicular, orbital, head
and neck, central nervous system, acoustic, pelvic,
respiratory tract, and urogenital), neurofibromatosis,
and cervical dysplasia.
The present invention is not limited to the
detection of circulating epithelial cells only.
Endothelial cells have been observed in the blood of
patients having a myocardial infarction. Eridothelial
cells, myocardial cells, and virally infected cells,
like epithelial cells, have cell type specific
determinants recognized by available monoclonal
antibodies. Accordingly, the methods and the kits of
the invention may be adapted to detect such circulating
endothelial cells. Additionally, the invention allows
for the detection of bacterial cell load in the
peripheral blood of patients with infectious disease,
who may also be assessed using the compositions, methods
and kits of the invention.
Several citations to journal articles, US Patents
and US Patent applications are provided hereinabove.
76
~......õ.,.,.,,_,,._..,,....,~-
...M.._,....n......__,......._._...,_...._..,_._._,...~,._.~......- a.
CA 02432363 2003-07-03
WO 99/41613 PCT/US99103073
While certain of the preferred embodiments of the
present invention have been described and specifically
exemplified above, it is not intended that the invention
be limited to such embodiments. Various modifications
may be made thereto without departing from the spirit of
the present invention, the full scope of which is
delineated in the following claims.
77
