Note: Descriptions are shown in the official language in which they were submitted.
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Title: Magnetic Enrichment of Circulating Cells, Fragments and Debris for
Enabling HTS Proteomics and Genomics in Disease Detection
Inventors: Gerald Doyle, Shawn Mark O'Hara, and Herman Rutner
PRIORITY INFORMATION
This application is a continuation-in-part of U.S. application 10/780,399,
filed 23 August 2002 as PCT/US02/26861, and US Provisional Applications
60/314,151, filed 23 August 2001, 60/369,628, filed 03 April 2002, and
60/524,918, filed 25 November 2003, under 35 USC 119(e). These
applications are incorporated by reference herein.
Background
Field of the Invention
This invention generally relates to the use of proteomic and mRNA transcript
investigation as diagnostic tools as it relates to the fields of oncology and
diagnostic testing. More specifically, the present invention relates to the
use
of proteomics and mRNA transcript profiling as a source of information in the
analysis of tumor cells for early diagnosis of cancer and in predicting
clinical
outcomes.
Background Art
Most cancer deaths are not caused by the primary tumor. Instead, death
results from metastases, i.e. multiple widespread tumor colonies established
by malignant cells that detach themselves from the site of the original tumor
and travel through the body 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. Because of the
difficulty in detection, metastatic colonies are harder to detect and
eliminate
and it is often impossible to treat all of them successfully. From a clinical
perspective, metastasis is considered a conclusive event in the natural
progression of cancer. Moreover, the ability to metastasize is the property
that uniquely characterizes a malignant tumor.
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Cancer metastasis comprises a complex series of sequential events.
These begin with the extension from the primary locus into surrounding
tissues, penetration into body cavities and vessels, release of tumor cells
for
transport through the circulatory system to distant sites, reinvasion of
tissue at
the site of arrest, and 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 vliell as many other "tumor' antigens, such as PSA, CA
15.3, CA 125, PSMA, CA27.29. These 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, one test has proven to be useful in the early detection of
cancer (i.e. 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 the very early detection of prostate cancer, at a time when
it is best treated.
Despite the success of PSA testing, the test is not totally acceptable. For
example, high levels of PSA do not always correlate with cancer not 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 befter
concordance between those patients with cancers that will metastasize and
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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 (CTC) 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 performing reverse transcriptase PCR. As of
this date, 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 (Gomella LG. J of Urology. 158:326-337 (1997)). It is noteworthy
that PCR is difficult to perform quantitatively, i.e. determine the 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 105 to
106 cells purified away from interfering red blood cells. This corresponds to
a
practical lower limit of sensitivity of one tumor cell per 0.1 ml of blood.
Hence,
there needs to be about 10 tumor cells in an ml of blood before a 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
complementation between PCR primers and target sequence can be lost.
A useful diagnostic test needs to be very sensitive and reliable. A blood
test developed to detect the presence of a single tumor cell in one ml of
blood
(as described in US 6,365,362) corresponds to the detection of 3000 - 4000
total cells in circulation, a number that establishes tumors in inoculated
animals. Further if 3000 - 4000 circulating cells represent 0.01% of the total
cells in a tumor, then it would contain about 4 x 107 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 could assess tumor burden. The general view is that tumors
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are initially well confined and hence there are few if any circulating cells
in
early stages of disease.
Based on the above, 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.
Many laboratory and clinical procedures employ biospecific 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 others.
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, such as by settling, or 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 biospecific affinity reactions (see, for example, US
4,554,088). 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.
Consequently, immunomagnetic separation as described in US 6,365,362
has significant clinical ramifications for the diagnosis and treatment of
cancer.
These include the fact that tumor cells are present in the blood of patients
who are considered to have clinically localized, primary tumors, that the
number of tumor cells present in the circulation correlates with all stages of
cancer from its inception to its terminal stages and that the changes in the
number of tumor cells present in the circulation is indicative of disease
progression. A decrease in the number of circulating tumor cells is indicative
of an improvement in patient status or efficacy of treatment, whereas an
increase indicates a worsening of the disease.
As shown in W000/47998, US #6,190,870, and other publications, CTC
can circulate as both live and dead cells, wherein "dead" comprises the full
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range of damaged and fragmented cells as well as CTC-derived debris. The
tumor burden is probably best represented by the total of both intact CTC,
including clusters, and damaged CTC, which bear morphological
characteristics of cells, but are distinct from clumps and/or aggregates.
However, some damaged cells, may have large pores allowing leakage of the
liquid and particulate cytosolic contents resulting in a change in the buoyant
densities from about 1.06-1.08 to greater than 1.12, or well above the
densities of RBC (live and dead cells can be separated at the interface of
gradients of d=1.12 and 1.16 according to a Pharmacia protocol).
Conventional density gradients, as used in # W000/47998 would lose such
damaged CTC in the discarded RBC layer having a range in density of about
1.08 to 1.11. CTC debris that is positively stained for cytokeratin may also
have densities falling in the RBC or higher ranges, since most intracellular
components (with the possible exception of lipophilic membrane fragments
that may be located near the plasma-buffy coat interface) have densities in
the range of 1.15 to 1.3. Hence, a substantial portion of damaged CTC and
CTC debris may be located outside the buffy coat layer, and would not be
seen by the density gradient methods, such as those in W000/47998. Some
images of damaged or fragmented CTC are shown, but it is quite possible the
damage occurred during cytospin or subsequent processing, and is thus
artifactual. While the densities of most intact tumor cells may fall in the
WBC
region, it is quite likely that damaged CTC in patient samples have higher
densities that may place them in the RBC layer; outside the reach of gradient
techniques.
US Patent Application 10/780,399 describes methods for binding
fragments and debris to beads. That application describes analysis of the
density of fragments and debris of interest. Upon centrifugation, the beads
will be located in a layer above RBC, because of the pre-determined specific
gravity (density) of the beads coupled to fragments and/or debris. However,
this system is dependent on correctly binding fragments and debris to these
beads. If any other sample component binds the beads, they may not appear
in the desired location, and subsequently will not be subject to analysis.
Epithelial cells in their tissue of origin obey established growth and
development "rules". Those rules include population control. This means that
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under normal circumstances the number and size of the cells remains
constant and changes only when necessary for normal growth and
development of the organism. Only the basal cells of the epithelium or
immortal cells will divide and they will do so when it is necessary for the
epithelium to perform its function, whatever it is depending in the nature and
location of the epithelium. Under some abnormal but benign circumstances,
cells will proliferate and the basal layer will divide more than usual,
causing
hyperplasia. Under some other abnormal but benign circumstances, cells
may increase in size beyond what is normal for the particular tissue, causing
cell gigantism, as in folic acid deficiency.
Epithelial tissue may increase in size or number of cells also due to pre-
malignant or malignant lesions. In these cases, changes similar to those
described above are accompanied by nuclear abnormalities ranging from mild
in low-grade intraepithelial lesions to severe in malignancies. It is believed
that changes in these cells may affect portions of the thickness of the
epithelium and as they increase in severity will comprise a thicker portion of
such epithelium. These cells do not obey restrictions of contact inhibition
and
continue growing without tissue controls. When the entire thickness of the
epithelium is affected by maiignant changes, the condition is recognized as a
carcinoma in situ (CIS).
The maiignant cells eventually are able to pass through the basement
membrane and invade the stroma of the organ as their malignant potential
increases. After invading the stroma, these cells are believed to have the
potential for reaching the blood vessels. Once they infiltrate the blood
vessels, cells find themselves in a completely different environment from the
one they originated.
The cells may infiltrate the blood vessels as single cells or as clusters of
two or more cells. A single cell of epithelial origin circulating through the
circulatory system is destined to have one of two outcomes. It may die or it
may survive.
Single Cells:
1. The cell may die either through apoptosis due to internal changes or
messages in the cell itself. These messages may have been in the cell
before intravasation or they may be received while in the blood, or it may
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die due to the influence of the immune system of the host, which may
recognize these cells as "alien" to this environment. The results of cellular
death are identifiable in imaging systems as enucleated cells, speckled
cells or amorphous cells. These cells do not have the potential for cell
division or for establishing colonies or metastases.
= Enucleated cells are the result of nuclear disintegration and
elimination (karyorrhexis and karyolysis). They are positive for
cytokeratin, and negative for nucleic acid.
= The speckled cells are positive for cytokeratin and DAPI and show
evidence of cellular degeneration and cytoplasmic disintegration.
These cells may represent response to therapy or to the host's
immune system as the cytoskeletal proteins retract.
= Another dying tumor cell identifiable is the amorphous cell. These
cells are probably damaged during the preparation process, a sign
that these may be weaker, more delicate cells but may also be the
result of apoptosis or immune attack.
2. A viable malignant epithelial cell may have the potential to survive the
circulation and form colonies in distant organs. These "survivor cells"
appear in as intact cells with high nuclear material/cytoplasmic material
ratio. These cells are probably undifferentiated and can potentially divide
in blood and form small clusters (Brandt et al. "Isolation of prostate-derived
single cells and cell clusters from human peripheral blood" Cancer
Research 56, 4556-4561, 1996) that may extravasate in a distant capillary,
where the cell may establish a new colony, or it may remain as a single
cell until it extravasates, dividing once it establishes itself in the new
tissue, starting this way a new colony.
Once a new colony is established in a new organ, some malignant cells
will continue replicating to form a new tumor. If they reach new capillaries,
the
metastasis story may be repeated and secondary metastasis occurs.
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Monitonng.
By monitoring during treatment in patients with known carcinomas, a
decrease in the number of tumor cells and/or some change in an appropriate
index may represent a response to patient therapy.
For example, the response index represents a measure of response to a
patient therapy whereby
= Total tumor cells = Dying cells + Survivor cells (TTC = DC + SC).
= Response Index = dying cells / total tumor cells (R1= DC / TTC).
Thus, the higher the response index, the better the response to
therapy. A low response index may indicate that the patient is not responding
to the treatment and or that the pt's immune system is not able to handle the
tumor load.
A patient who has 50 total tumor cells that were all survivor cells at pre-
treatment visit (a RI = 0/50 = 0) and has 50 TTC on follow-up (after
treatment)
visit may have different outcomes depending in the RI. If all the TTC are SC
(i.e. DC = 0), there was no response to therapy. If there are 50 cells but the
response index is 40/50 = 0.8, then either the immune system or the therapy
is having a negative effect on tumor load, therefore, is a positive patient
response.
Follow-up
When a pap smear is diagnosed as having cells with atypia or low-grade
intraepithelial lesions, there is always the possibility that these patients
have a
more severe abnormality, which cells were missed as a sampling error.
These patients are biopsied and asked to return in three months for a repeat
pap smear. If the atypical cells were concurrent with a small focal area of
malignant cells that did not get sampled, the patient will wait 3 months
before
she gets any follow-up, opening the possibility of misdiagnosis. Using image
analysis all patients with an abnormal pap (5-10% of the pap smears in the
USA) are relatively easily and quickly tested for circulating epithelial
cells.
Patients with positive tests can be followed-up aggressively. This simplifies
the decision making process for the physician and health professionals.
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Screening
Image cytometry analysis is useful for screening the general population.
Identification of CTC in a patient could indicate that there is a primary
malignancy that has started or is starting the process of metastasis. If these
cells are identified as of the tissue of origin with new markers, then organ
specific tests, like guided fine needle aspirations (FNA) can be used to
verify
the presence or absence of such malignancies. Patients where a primary
cannot be identified can be followed-up with repeat tests after establishing
an
individual base line.
All or some of the above-cited factors were found to contribute to debris
and/or aggregate formation that have been observed to confound the
detection of CTC by direct enrichment procedures from whole blood as
disclosed in this invention. The number of intact CTC, damaged or suspect
CTC as well as the degree of damage to the CTC, may further serve as
diagnostically important indicators of the tumor burden, the proliferative
potential of the tumor cells and/or the effectiveness of therapy. The present
invention has a distinct advantage in that the methods and protocols of the
prior art combine unavoidable in vivo damage to CTC with avoidable in vitro
storage and processing damage, thus yielding erroneous information on CTC
and tumor burdens in cancer patients. This relatively simple blood test
described herein, which functions with a high degree of sensitivity and
specificity, can be thought of as a "whole body biopsy".
Proteomics
Incorporating a more global analysis of diagnosis, follow-up, and
screening as related to protein expression is another embodiment of the
present invention. Assessing global patterns of protein expression in
individual cells, tissues, or body fluids, has been the basic foundation in
proteomics and provide an improvement to current methods. Coupled with
genetic information, protein expression in individual cells can take on
several
different forms based upon the nucleotide sequence, whether a splice variant
occurs, or whether there is a post-translational modification. Thus, the
transcription, translation, and post-translational modification of each
protein
define a specific biochemical function within a living cell. Proteomics looks
at
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the transcripts of genomic DNA (messenger RNA) as they directly encode
proteins, and that these proteins are further modified by mechanisms such as
phosphorylation or glycosylation. As a consequence of this sequence of
events, there are functional variations in protein expression.
Thus, proteomics is a process of transcriptional profiling to determine
which genes, or combination thereof, are transcribed in a particular cell type
or disease state. To this end, protein profiling is examined by various
techniques which include two-dimensional gel electrophoresis (2D-Gel) and
mass spectroscopy (MS), co-immunoprecipation, affinity chromatography,
protein binding analysis, overlay analysis, using yeast in protein-protein
interaction, the analysis of signal transduction and other complex cellular
process, three-dimensional structure modeling and large-scale protein folding,
and the incorporation of bioinformatics with proteomic data.
Two-dimensional gel electrophoresis alone has several inherent problems,
especially when applied in diagnosis. These include difficulties in the
analysis
of the gels, the insufficiency of the resolving power to separate various
distinct
proteins in a particular sample, and a lack of reproducibility from one gel
sample to the next.
Methods are available for utilizing MS in the analysis of target
polypeptides. Here, the polypeptides are solubilized in a solution or reagent
system depending upon the properties of the polypeptide (i.e. organic or
inorganic solvents) and the type of MS performed (WO 93/24834 by Chait et
al.).
Mass spectrometer analysis includes ionization (I) techniques, including
but not limited to matrix assisted laser desorption (MALDI), continuous or
pulsed electrospray (ESI) and related methods (IONSPRAY or
THERMOSPRAY), or massive cluster impact (CI). These ion sources are
matched with detection formats including linear or non-linear reflection time-
off-light (TOF), single or multiple quadropole, single or multiple magnetic
sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, LC/MS,
MS/MS, and combinations thereof.
Matrix-assisted laser desorption/ionization time of flight mass spectrometry
(MALDI-TOF MS) refers to the formation of a matrix with several small, acidic,
light absorbing chemicals that is mixed in solution with the analyte in such a
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manner so that, upon drying on the probe element, the crystalline matrix-
embedded analyte molecules are successfully desorbed (by laser irradiation)
and ionized from the solid phase (crystals) into the gaseous or vapor phase
and acceierated as intact molecular ions. In the MALDI process, the analyte
is mixed with a freshly prepared solution of the chemical matrix and placed on
the inert probe element surface to air dry just before the mass spectrometric
analysis (see US 5,808,300).
Another general category, utilizing a sample presenting means, is
Surfaces Enhanced for Laser Desorption/Ionization (SELDI) and described in
US 6,020,208, within which there are three (3) separate subcategories. The
SELDI process is directed toward a sample presenting means (i.e., probe
element surface) with surface-associated (or surface-bound) molecules to
promote the attachment and subsequent detachment of analyte molecules in
a light-dependent manner, wherein the surface-associated molecule(s) are
selected from the group consisting of photoactive (photo labile) molecules
that
participate in the binding (docking, tethering, orcross linking) of the
analyte
molecules to the sample presenting means (by covalent attachment
mechanisms or otherwise).
Regardless of the MS method, the mass of the target polypeptideis then
compared to the mass of a reference polypeptide of known identity.
MS based processes for detecting a particular nucleic acid sequence in a
biological sample has been described in US 6,043,031. The process is used
to diagnose a genetic disease or chromosomal abnormality, a predisposition
to a disease or condition, infection by a pathogenic organism, or for
determining heredity. Detection of the desired fragments is optimum between
7,000 to 20,000 Da obtained from tryptic digests.
The use of proteomics in diagnosing the existence or predicting the
development and/or progression of abnormal physiological conditions based
upon the presence of proteomic materials has been previously described (US
20020260420). Several recent publications and reviews by L. Anderson of
The Plasma Proteome Institute (Washington, DC) also discuss the status of
current MS methods in proteomics and the requirements for adapting highly
sophisticated MS methods to practical clinical diagnostics, for example,
detection of less than 20 relevant protein markers, cost per analysis of $2-
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$100 and assay time of about 15 minutes. After obtaining a patient sample
containing proteomic materials, the patient sample is prepared by isolating
proteomic material with characteristics identifiable for normal and abnormal
physiological conditions or associated predictive endpoints, e.g down
regulation or up regulation of proteins also present in healthy individuals.
The
proteomic materials are separated to permit analysis of one or more specific
proteomic materials thereby enabling the diagnostician to characterize an
individual's condition as being either positively or negatively indicative of
one
or more abnormal physiological conditions.
While proteomics and current methods have been applied in cancer
diagnostics, such methods lack simple and efficient S/N amplification or pre-
enrichment methods that would improve the sensitivity and reduce the sample
processing time and cost of analysis of clinical specimens.
Summary of the Invention
The present invention provides a tool for clinicians in the diagnosis and
prognosis of disease states such as cardiovascuiar disorders and cancer, and
provides a sensitive, simple, and efficient analysis of disease detection to
complement other means of detection known in the art.
The methods and reagents described in this invention are used to analyze
circulating tumor cells, clusters, fragments, and debris. Analysis is
performed
with a number of platforms, including flow cytometry and imaging systems and
mRNA transcript profiling. The examples show the importance of not only
analyzing obvious or intact CTC, but suspect CTC or damaged fragments,
clusters of CTC, and debris. Similar analysis is possible with endothelial
cells.
In this type analysis, assessing the damage that forms fragments and debris
is easier. It is also possible to inhibit further damage of CTC between the
blood draw and sample processing through the use of stabilizing agents.
It has been shown herein that the ability to differentiate between in vitro
damage, caused by specimen acquisition, transport, storage, processing, or
analysis, and in vivo damage, caused by apoptosis, necrosis, or the patient's
immune system. Indeed, it is desirable to confine, reduce, eliminate, or at
least qualify in vitro damage to prevent it from interfering in analysis.
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After amplification by immunomagnetic enrichment of cells and/or
fragments from a patient blood sample, an analysis of proteomic parameters
alone or in combination with cytometric imaging of circulating debris or cells
after immunomagnetic partitioning. Thus, 2-D Gel electrophoresis, MS,
SELDI or microarray detection of cells and fragments would be used alone or
in conjuction with image analysis on the enriched fraction of debris and/or
cells, captured by positive selection of antibody-coupled magnetic particles.
The present invention also includes any specific antibody-antigen, ligand-
receptor, or labeling means. MS is accomplished directly on the captured
ferrofluid particles or on the captured target materials after dissociation
from
the ferrofluid by a reversible binding reaction, such as by the dissociation
of
the bond between target-Mab-desthiobiotin and streptavidin on the ferrofluid
with soluble biotin to liberate the Mab labeled target material. The direct
mode is most suited for diagnostic correlation with cell counts, clinical
diagnosis and the ability to differentiate target material from ferrofiuid
associated proteins, as well as potential utility as a complementary or
independent modality to cell imaging.
A second approach is to limit analysis to only MS after immunomagnetic
enrichment (or non-magnetic enrichment) from separate, unprocessed
specimens such as whole blood, plasma or serum. This approach is without
cell permeabilization, antibodies and staining reagents, incorporated with
image analysis, to minimize the introduction of extraneous components that
would interfere with MS analysis.
The one embodiment of the present invention is the enrichment of target
specific cell fragments, debris, and non-particulate soluble protein. These
include immune complexes which are normally present at low levels in the
early stages of disease and increase as the disease progresses.
Incorporating proteomics in cancer detection, especially at early stages,
provides additional information in the analysis of circulating rare cells if
enrichment provides sufficient mass for MS detection. In addition to a
substantial amount of CTC debris present during low CTC, capture of debris
containing the same surface markers as the intact cells, followed by MS
analysis provides a new platform for early cancer diagnosis.
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In addition with the use of monclonal antibodies as capture agents (i.e. CD
146, CD 105, CD 31, CD 133, CD 106), the present invention considers
diseases associated with circulating endothelial cells and their analysis.
These diseases include those relating to cardiovascular disorders.
Herein are described methods to diagnose, monitor, and screen disease
based on circulating rare cells, including malignancy as determined by CTC,
clusters, fragments, and debris. Also, proteomic and transcriptome analysis,
especially with the enriched cell/cell debris/cell fragment components, are
utilized in methodologies for diagnosing, monitoring and screening disease.
Brief Description of the Drawings
Figure 1: Models of tumor shedding and metastasis. Ia. shows possible
stages of cells, clusters, and fragments. 1 b. shows the same model with
actual images from samples.
Figure 2: Flow cytometric analysis of immunomagnetically enriched tumor
cells from a 7.5m1 blood of a metastatic prostate patient.
Figure 3: Image cytometry analysis with 7.5m1 blood sample from a
metastatic prostate cancer patient that was immunomagnetically enriched for
tumor cells. The lines of thumbnails correspond to the different dyes used in
the staining process showing tumor candidates stained with cytokeratin PE
and DAPI.
Figure 4: Classifications of tumor cells from a whole blood sample of a
patient with metastatic prostate cancer stained with cytokeratin PE and DAPI.
A: intact cells B: damaged tumor cells C: tumor cell fragments.
Figure 5: A comparison of the number of obvious CTC and suspect CTC in
20 clinical samples.
Figure 6: Classification of paclitaxel treated LnCaP cells spiked into whole
blood and isolated then stained with cytokeratin PE and DAPI. A: intact cells
B: dying tumor cells C: tumor cell fragments
Figure 7: Outline of one embodiment in a sample preparation for proteomic
analysis.
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Detailed Description of the Invention
General Definitions
Proteomics refers to the study of proteins and their DNA messenger RNA
transcripts that directly encode for them. These expressed proteins can be
further modified by post-translational modification, e.g. such as
phosphorylation and glycosylation that alter protein expression.
The term "rare cells" as used herein refers to a variety of cells,
microorganisms, bacteria, and the like. Cells are characterized as rare in a
sample because they are not present in normal samples of the same origin,
and are several orders of magnitude lower in concentration that the typical
cells in a normal sample. Embodiments of the present invention include
circulating cancer cells, virally, infected cells, fetal cells in maternal
circulation,
or endothelial cells efficiently isolated from non-rare cells and other
bioentities, using the methods and apparatus of the present invention in
conjunction with previously described technology (US 6,365,362).
The term "analyte" refers to any atom and/or molecule; including their
complexes and fragments ions. In the case of biological
molecules/macromolecules or "biopolymers", such analytes include but are
not limited to: proteins, peptides, DNA, RNA, carbohydrates, steroids, and
lipids.
Detailed Description
Evidence that minimal residual disease in patients with carcinoma has
clinical significance is mounting. To effectively monitor minimal residual
disease, a qualitative and quantitative assessment is needed. As the
frequency of carcinoma cells in blood or bone marrow is low, the laborious
manual sample preparation methods involved in the preparation of samples
for analysis often leads to erroneous results. To overcome these limitations a
semi-automated sample preparation system was developed that minimize
variability and provide more consistent results, as described in commonly-
owned US Application No. 10/081,996 (filed 20 February 2002) which is
incorporated by reference herein.
Various methods are available for analyzing or separating the above-
mentioned target substances based upon complex formation between the
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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, by centrifugation of
finely
divided particles or beads coupled to the target substance. 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. Generally, any material that
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 of size; large (1.5 to
about 50 microns), small (0.7-1.5 microns), or colloidal (<200nm), which are
also referred to as nanoparticles. Nanoparticles, also known as ferrofluids or
ferrofluid-like materials, have many of the properties of classical
ferrofluids,
and 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.
The efficiency with which magnetic separations depends on many factors.
For example, 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, reflecting poorly on the efficiency.
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 Dynal beads, cells are labeled through collisions created
by
mixing of the system, as the beads are too large to diffuse effectively. Thus,
if
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a cell were present in a population at a frequency of 1 cell per mi 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 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
as possible. Increasing the concentration of the larger particles does not
markedly improve the ability to enumerate the cells of interest or to examine
them.
The preferred magnetic particles for use in the present invention are
particles that behave as colloids. Such particles are characterized by their
sub-micron particle size, which is generally less than about 200nm, and their
stability to gravitational separation from solution for extended periods of
time.
In addition to the many other advantages, this size range makes individual
particles essentially invisible to analytical techniques commonly applied to
cell
analysis. Particles within the range of 90-150nm 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 magnetic 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 carbohydrates such as sialic acid
residues on the surface of non-target cells, lectins, glycproteins, and other
membrane components. In addition, the material should contain as much
magnetic.mass per 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 nanoparticies is sufficiently small
such that their Brownian energy exceeds their magnetic moment. As a
consequence, magnetic alignment and subsequent mutual attraction/repulsion
of these colloidal magnetic particles does not appear to occur even in
moderately strong magnetic fields, contributing to solution stability.
Finally,
the magnetic particles are separated in high magnetic gradient external field
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separators, facilitating sample handling and providing 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 4,795,698, 5,597,531, and 5,698,271, each
incorporated by reference herein.
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 label rare events, such as tumor cells in blood. Additionally for
magnetic separations to be successful, the magnetic particles must be
specific for epitopes that are not present on hematopoetic cells.
A large variety of analytical methods and criteria are used to identify tumor
cells, and the first attempfis are being undertaken to standardize criteria
that
define what constitutes a tumor cell by immunocytochemistry. In this study,
blood samples from prostate cancer patients were immunomagnetically
enriched for cells that expressed EpCAM. Tumor cells were identified by the
expression of the cytoskeletal proteins cytokeratin (CK+), the absence of the
common leukocyte antigen CD45 (CD45-) and the presence of nucleic acids
(NA+) by multicolor fluorescence analysis. Rare events or rare cells can be
immunophenotyped by both flowcytometry and fluorescence microscopy.
Flowcytometric analysis excels in its ability to reproducibly quantify even
low
levels of fluorescence whereas microscopy has the better specificity as
morphological features can aid in the classification of the
immunophenotypically identified objects. Although there was a correlation
between the number of CTC detected in blood of prostate cancer patients by
flowcytometry and microscopy, microscopic examination of the CK+, CD45-,
NA+ objects showed that only few of the objects appeared as intact cells.
This observation agrees with other reports that showed apoptosis in a
substantial portion of circulating tumor cells.
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The terms "biological specimen" or "biological sample" may be used
interchangeably, and refer to a small potion of fluid or tissue taken from a
human test subject that is suspected to contain cells of interest, and is to
be
analyzed. A biological specimen refers to the fluidic portion, the cellular
portion, and the portion containing soluble material. Biological specimens or
biological samples include, without limit bodily fluids, such as peripheral
blood, tissue homogenates, nipple aspirates, colonic lavage, sputum,
bronchial (alveolar) lavage, pleural fluids, peritoneal fluids, pericardial
fluids,
urine, and any other source of cells that is obtainable from a human test
subject. An exemplary tissue homogenate may be obtained from the sentinel
node in a breast cancer patient.
The term "rare cells" is defined herein as cells that are not normally
present in biological specimens, but may be present as an indicator of an
abnormal condition, such as infectious disease, chronic disease, injury, or
pregnancy. Rare cells also refer to cells that may be normally present in
biological specimens, but are present with a frequency several orders of
magnitude less than cells typically present in a normal biological specimen.
The term "determinant", when used in reference to any of the foregoing
target bioentities, refers broadly to chemical mosaics present on
macromolecular antigens that often induce an immune response.
Determinants may also be used interchangeably with "epitopes". A
"biospecific ligand" or a "biospecific reagent," used interchangeably herein,
may specifically bind determinants. A determinant refers to that portion of
the
target bioentity involved in, and responsible for, selective binding to a
specific
binding substance (such as a ligand or reagent), 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
agents, ligands and/or reagents having binding affinity therefor, 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.
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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, 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). Analysis can be performed using
any of a number of commonly used platforms, including multiparameter flow
cytometry, immunofluorescent microscopy, laser scanning cytometry, bright
field base image analysis, capillary volumetry, spectral imaging analysis,
manual cell analysis, image cytometry analysis, and other automated cell
analysis.
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 phrase "early stage cancer" is used interchangeably herein with
"Stage I" or "Stage II" cancer and refers to those cancers that 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 process of substantially
increasing the ratio of target bioentities (e.g., tumor cells) to non-target
materials in the processed analytical sample compared to the ratio in the
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original 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 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 terms "anti-coagulant" or "anti-coagulating agent" may be used
interchangeably, and refer to compositions that are added to biological
specimens for the purpose of inhibiting any undesired natural or artificial
coagulation. An example of coagulation is blood clotting and common anti-
coagulants are chelating agents, exemplified by ethylenediamine tetraacetic
acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,2-
diaminocyclohexane tetraacetic acid (DCTA), ethylenebis(oxyethylenenitrilo)
tetraacetic acid (EGTA), or by complexing agents, such as heparin, and
heparin species, such as heparin sulfate and low-molecular weight heparins.
This may be further collectively defined as "clumping' or "clump formation".
However, such clumps must be differentiated from "clusters" or aggregates of
CTC that are counted as a single Intact CTC if they meet the classification
criteria for Intact CTC.
Clusters of CTC are believed to have greater proliferative potential than
single CTC and their presence is thus diagnostically highly significant. One
possible cause for an increased propensity to establish secondary metastatic
tumor sites may be the virtue of their adhesiveness. An even more likely
cause is the actual size of a CTC cluster; larger clusters will become lodged
in
small diameter capillaries or pores in bone. Once there, the viability of the
cells in the cluster would determine the chance of survivability at the new
metastatic site.
The ideal "stabilizer" or "preservative" (herein used interchangeably) is
defined as a composition capable of preserving target cells of interest
present
in a biological specimen, while minimizing the formation of interfering
aggregates and cellular debris in the biological specimen, which in any way
can impede the isolation, detection, and enumeration of targets cells, and
their differentiation from non-target cells. In other words, when combined
with
an anti-coagulating agent, a stabilizing agent should not counteract the anti-
coagulating agent's performance. Conversely, the anti-coagulating agent
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should not interfere with the performance of the stabilizing agent.
Additionally, the disclosed stabilizers also serve a third function of fixing,
and
thereby stabilizing, permeabilized cells, wherein the expressions
"permeabilized" or "permeabilization" and "fixing", "fixed" or "fixation" are
used
as conventionally defined in cell biology. The description of stabilizing
agents
herein implies using these agents at appropriate concentrations or amounts,
which would be readily apparent to one skilled in cell biology, where the
concentration or amount is effective to stabilize the target cells without
causing damage. One using the compositions, methods, and apparatus of
this invention for the purpose of preserving rare cells would obviously not
use
them in ways to damage or destroy these same rare cells, and would
therefore inherently select appropriate concentrations or amounts. For
example, the formaldehyde donor imidazolidinyl urea has been found to be
effective at a preferred concentration of 0.1-10%, more preferably at 0.5-5%
and most preferably at about 1-3% of the volume of said specimen. An
additional agent, such as polyethylene glycol has also been found to be
effective, when added at a preferred concentration of about 0.1% to about
5%, more preferably about 0.1% to about 1%, and most preferably about
0.1 % to about 0.5% of the specimen volume.
Stabilizing agents are necessary to discriminate between in vivo tumor cell
disintegration and disintegration due to in vitro sample degradation.
Therefore, stabilizing agent compositions, as well as methods and apparatus
for their use, are described in a co-pending application entitled
"Stabilization
of cells and biological specimens for analysis." That commonly owned
application is incorporated by reference herein.
The terms "obvious cells" or "intact cells" may be used interchangeably,
and refer to cells found during imaging analysis that contain nucleic acid and
cytokeratin. These cells are usually visually round or oval, but may
sometimes be polygonal or elongated, and appear as individual cells or
clusters of cells. The nucleic acid area (i.e. labeled by nucleic acid dye) is
smaller than the cytoplasmic area (i.e. labeled by anti-cytokeratin), and is
surrounded by the cytoplasmic area.
The terms "suspicious cells", "suspect cells", or "fragments" may be used
interchangeably, and refer to cells found during imaging analysis that
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resemble intact cells, but are not as visually distinct as intact cells. Based
on
imaging analysis, there are a number of possible types of suspect cells,
including:
1. Enucleated cells, which are shaped like Obvious cells, are positively
stained for cytokeratin, but negative for nucleic acid;
2. Speckled or punctate cells, which are positively stained for nucleic
acid, but have irregularly-stained cytokeratin; and
3. Amorphic cells, which stain positively for cytokeratin and nucleic acid,
but are irregular in shape, or unusually large.
These suspicious cells are considered in the present invention because
they give additional information to the nature of the CTC, as well as the
patient's disease. The staining or image artifacts observed during analysis
provide additional informaton. For example, enucleated cells sometimes
appear to have a "ghost" region where the nucleus should have stained, but
the corresponding region is nucleic acid negative. This may be caused by a
number of external factors, including the labeling or imaging techniques.
Also, cells have been observed with "detached" nuclei. While this may
possibly indicate a cell releasing its nucleus, it is more likely that this
appears
due to an artifact of the imaging system. However, such "artifacts," when
real,
give valuable information about what may be happening to the intact cells.
Therefore, the present invention considers suspicious cells as a component in
the analysis.
Cell fragments are different than "debris" in that debris does not
necessarily resemble a cell. The term debris as used herein, refers to
unclassified objects that are specifically or non-specifically labeled during
processing, and are visible as images during analysis, but are distinct from
intact and/or suspect cells. For example, it has been observed that damaged
cells will release nuclear material. During processing, this nuclear material
may be non-specifically magnetically labeled, and subsequently labeled with
the nucleic acid stain. During analysis, the magnetically labeled and stained
nuclear material can be observed when it has cytokeratin still attached. There
are other objects that are similarly magnetically selected and stained which
appear during analysis that are classified as debris.
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The term "morphological analysis" as used herein, refers to visually
observable characteristics for an object, such as size, shape, or the
presence/absence of certain features. In order to visualize morphological
features, an object is typically non-specifically stained. The term
"epitopical
analysis" as used herein, refers to observations made on objects that have
been labeled for certain epitopes. In order to visualize epitopic features, an
object is specifically stained or labeled. Morphological analysis may be
combined with epitopical analysis to provide a more complete analysis of an
object. ,
Figure 1 is a model of various CTC stages, including shedding and
metastasis. Figure 1 a. shows these stages for cells, clusters, fragments, and
debris. Figure 1 b. shows actual images from samples at these same stages.
The images of cells clusters, fragments, and debris were taken from patient
samples after immunomagnetic enrichment and image cytometry. The
images of tissue samples (Origin and Metastatic sites) were taken from
elsewhere (Manual of Cytology, American Society of Clinical Pathologists
Press. 1983).
Briefly, a single cell shed from a primary tumor into the blood either
survives or dies in blood. If it survives, it may possibly divide in blood, or
colonize at a secondary site. If the cell dies, depending on the method, the
cell degrades into various types of fragments or debris. Another possibility
is
a cluster of cells is shed from a primary tumor into the blood, where it may
dissociate into single cells, or remain intact, and colonize at a secondary
site.
If the cluster dissociates, it can behave similar to the single cell described
above. If the cluster remains intact, it is more likely to for a secondary
colony
for the reasons described above, which includes the large diameter cluster
becoming lodged in a small diameter capillary. Once lodged, if the cells are
viable, the cluster would form a new tumor.
The presence of fragments and debris with very few intact cells suggests
that there will be little chance of metastasis. Fragmented cells will not
divide,
and cannot form secondary tumors. Indeed, only intact CTC or possibly CTC
clusters would be capable of colonizing secondary sites. Identification of
antigens that play a role in the adhesion and penetration process may help.
Follow up and assessment of metastatic sites of the patients with and without
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clusters will also provide further insight. Nuclear morphology is used to
determine the activity status and abnormality of a cell. Chromatin clumping,
the presence or absence of nucleoli, and hyperchromasia, are criteria used to
determine whether a cell is benign or malignant, reacting to an immune
response, or reacting to treatment. The cytoplasmic morphology is used to
determine the level of differentiation (i.e. tissue of origin). For example,
cytomplasmic morphology can classify cells as squamous versus glandular.
During blood draw and subsequent specimen processing, the surviving
battered tumor cells present in the peripheral circulation may be further
stressed and damaged by turbulence during blood draw into an evacuated
tube and by specimen processing, e.g. transport of the blood tube and mixing
prior to analysis. Such mechanical damage is additional to on-going
immunological, apoptotic, and necrotic processes leading to destruction of
CTC that occur in vitro in a time dependent manner. We have found that the
longer the specimen is stored, the greater the loss of CTC, and the larger the
amounts of interfering debris and/or aggregates. Indeed, data presented in
this specification (Figures 2 and 3) show dramatic declines in CTC counts in
several blood specimens stored at room temperature for 24 hrs or longer,
indicating substantial in vitro destruction of CTC after blood draw. While the
losses of hematopoietic cells are well known phenomena and the subject of
above-cited patents by Streck Labs and by others, the occurrence of
mechanical damage due to mixing or transport have to date not been
recognized factors in the loss of CTC or rare cells. The formation of cellular
debris and the interfering effects of accumulating debris and/or aggregates in
the analysis of CTC or other rare cells have similarly been unrecognized to
date. It appears to be most evident and problematic in highly sensitive
enrichment assays requiring processing of relatively large blood volumes (5-
50mL), and subsequent microscopic detection or imaging of target cells after
volume reduction (less than 1 mL). Such debris are either not normally seen,
or do not interfere in conventional non-enrichment assays, for example, by
flow cytometry or in enrichment by density gradients methods.
To explore if these damaged epithelial cells and epithelial cell fragments
observed in patients could be caused by apoptosis of tumor cells induced by
chemotherapy, a model to mimic tumor cell death was developed. Cells of
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the prostate cell line LnCaP were cultured with or without paclitaxel and
spiked into blood of healthy donors. The immunomagnetically selected cells
of the paclitaxel treated samples resembled those observed in the patient
blood samples. Cells treated with paclitaxel displayed signs of apoptosis.
The punctate cytokeratin staining pattern of the cells appear to correspond
with a collapse of the cytoskeletal proteins. The initiating event in the
sequence resulting from the microtubule stabilizing effects of paclitaxel
which
in turn may activate the pro-apoptotic gene Bim that senses cytoskeletal
distress. Further evidence of caspase-cleaved cytokeratin resulting from
apoptosis was obtained with the M30 Cytodeath antibody (Roche Applied
Science, Mannheim, Germany) that recognizes an epitope of cytokeratin 18
that is only exposed following caspase cleavage in early apoptosis. Only the
paclitaxel treated LnCaP cells stained with M30 and most of the dimmer
cytokeratin cells stained with M30, which is consistent with cells undergoing
apoptosis.
With the technological resolution power of proteomics in recent years, the
use as tool for clinicians in diagnosis and prognosis is becoming practical.
The present invention utilizes this approach to provide clues in the early
diagnosis of cancer and in prediction of clinical outcomes.
One of the biggest problems in the clinical use of this approach is the
selective extraction or enrichment of the desired global target entities,
which
typically number fewer than 100, from highly complex samples containing
millions of irrelevant entities.
Prior attempts have used partial fractionation and enrichment on 2-D
electrophoresis. This is a highly tedious, inefficient and costly procedure,
resulting in a complex array of protein zones. The zones frequently contain
numerous components of identical size and charge/mass ratio, totally
unrelated to their functionality or diagnostic relevance. The analysis of
proteins in these zones becomes more completed after proteolytic digestion
into smaller fragments by mass spectrometry (MS). As mentioned above, MS
is a highly sensitive analytical tool for identification of mass fragments
based
on charge/mass ratios which allows reconstruction of the parent molecule, but
identification can only occur following removal of unrelated components.
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To increase sensitivity and remove these unrelated components, Surfaces
Enhanced for Laser Desorption/Ionization Time-Of-Fight (SELDI-TOF) MS on
multiplexed microarray, microchip, or biochip detection of specific target
proteins or protein digests are used on enriched fractions to provide an
analytical mechanism for diagnosis. A direct analysis of unpurified samples
such as serum and tissues by SELDI-TOF have been used by others, but
these require analysis of complex fragmentation profiles and associated large
data filed due to the presence of huge amounts of non-target materials.
In the case of rare target materials, effective detection requires prior
enrichment and current fractionation methods of two-dimensional
electrophoresis. As a diagnostic tool, these steps are affected by cost and
clinical utility. Using magnetic or non-magnetic enrichment as described in
the present invention reduces these unwanted factors and provides a basis
for developing MS as a diagnostic tool.
Besides magnetic separation, non-magnetic affinity-based solid phase
separation can also be used to selectively enrich specific targets or target
populations (e.g. antibody coated particles or solid phases for capturing the
target materials, followed by analysis of the enriched fraction without or
with
prior dissociation from the support).
Magnetic separation with ferrofluid particles described in US 6,365,362
provides a means of enrichment that is inexpensive and simple. Further,
these ferrofluid particles provide higher binding capacities than other larger
particles or non-magnetic solid phase particles (e.g. gel particles).
Thus, instead of running multiple costly and time-consuming MS analysis
on numerous non-target spots for two-dimensional electrophoresis, only one
single MS analysis on essentially target-specific proteins will be needed per
sample, thereby dramatically increasing throughput, sensitivity and
specificity
of detection.
The one embodiment of the present invention, in part, uses the procedure
described in US 6, 365,362 to incorporate multiparametric image cytometry
and morphological characterization of selectively stained tumor cells together
with proteomic analysis in cancer diagnosis. As previously described for rare
cell detection methods in cancer diagnosis and management, magnetic
enrichment of rare target cells, along with associated cell fragments and
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debris are coupled with proteomics as an alternative means of cancer cell
detection.
Magnetic enrichment of rare target cells can occur after pretreatment with
or without preservative (U.S. application 10/780,399). After immunomagnetic
(or alternatively non-magnetic) enrichment, pathological cells, cell
fragments,
debris, and soluble cell fractions from patient specimens are assessed by MS,
SELDI, microchips, biochips, or multiplexed micro array analysis. The
detection of cell fragments, debris and soluble cell fractions from patient
specimens are found in large quantities in the blood or tissues of some cancer
patients, allowing for MS analysis. The importance and potential diagnostic
utility of cell debris detection has been the subject of pending U.S.
application
10/780,399. Figure 6 shows a diagramatic representation of one method for
isolating the debrilcell fraction. The components of the crude enriched whole
blood fraction are separated by acidification to remove bovine se,rum
ferrofluid
(BSA-FF) and streptavidin, conjugated to a monoclonal antibody (streptavidin-
Mab). White blood cells (WBC) and red cells in the remaining cell and cell
debris are removed by negative selection. Lipids, such as found in the
membrane, are removed by solvent extraction. Thus, the only remaining
components are the rare cells of interest (i.e. tumor cells and/or endothelial
cells) and serum protein/glycoproteins. These are enriched by N2
evaporation.
When imaging and proteomics are combined in this format, the
magnetically enriched fractions are retrieved from the viewing chamber after
imaging by magnetic separation of the supernatant buffer and buffer
components. The buffer is replaced by an enzyme-compatible saline solution
and analyzed directly. Instead of direct analysis, reversible chemical
dissociation or tryptic dissociation digestion into fragments prior to MS
analysis are done within the chamber by adding a dissociating agent or
enzyme solution to a suspension of the magnetic particles to separate the
ferrofluid particles. Thus, the captured cell and/or proteins are dissociated
from the ferrofluid particles with an optional digestion to peptide fragments
prior to analysis by MS. The preferred size for MS detection after tryptic
digestion is 7,000 to 20,000 Da. This is a range that is lower than the sizes
of
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most soluble tumor markers, and much lower than the sizes reported for
circulating tumor cell debris.
Another embodiment incorporates magnetic enrichment of the target cells
and/or cell debris using a proteomic analysis system as the only platform.
Immunomagnetic enrichment provides a simple amplification method to
improve the sensitivity to a level that allows for consistent diagnostic use.
As a specific example, the captured target cells or proteins, complexed
with desthiobiotinylated monoclonal antibody-ferrofluid (Mab-FF), are
assessed by MS either alone or in combination with image analysis. For MS
analysis, the captured target cells or proteins are dissociated from the
ferrofluid with biotin to generate and enriched sample fraction, free of
proteins
derived from the ferrofluid particles. For cell and/or proteins from
epithelial-
derived cell membranes of circulating tumor cells (and debris from damaged
circulating tumor cells), epithelial cell adhesion molecule (EpCAM) MAb-FF
captures most of the target entities in the enriched sample fraction while
other
gradient methods may lose a substantial portion of entities.
Both approaches yield tumor specific mass profiles that are subtracted
from MS profiles for BSA, MAbs-FF, or other sample enriched components.
These subtracted profiles can be compared for disease and/or disease state,
yet without knowledge of the identity of the measured proteins. The two
embodiments, mentioned above, allow for complementary confirmation of
CTC obtained by imaging, or possibly earlier cancer diagnosis in MS analysis
without associated imaging. Surprisingly, MS analysis can provide a means
for early cancer diagnosis even before intact CTC are detectable by imaging
from a small blood specimen. For example, Her2/neu levels in the low ng/ml
range in plasma can be immunomagnetically enriched to provide debris levels
sensitive enough for MS analysis.
Further with respect to immunomagnetic enrichment and multiparametric
image cytometry, MS proteomic analysis would obviate the need for
immediate analysis or stabilization of blood samples for later analysis,
required in image analysis. In addition, controlled aggregation may be
unnecessary when analyzing captured debris. These factors could provide an
improved sensitivity to diseases such as early cancer detection.
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It should be noted that a number of different cell analysis platforms can be
used to identify and enumerate cells in the enriched samples. Examples of
such analytical platforms are described in US Patents 5,876,593; 5,985,153
and 6,136,182, each of which are incorporated by reference herein as
disclosing the respective apparatus and methods for manual or automated
quantitative and qualitative cell analysis.
Other analysis platforms include laser scanning Cytometry (Compucyte),
bright field base image analysis (Chromavision), and capillary Volumetry
(Biometric Imaging).
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. The immunomagnetic sample
preparation is important for reducing sample volume and obtaining as much
as a 104 fold enrichment of the target (epithelial) cells. The reagents used
for
the multi-parameter flow cytometric analysis are optimized such that
epithelial
cells are located in a unique position in the multidimensional space created
by
the listmode acquisition of two light scatter and three fluorescence
parameters. These include
1. an antibody against the pan-leukocyte antigen, CD45 to identify
leucocytes (non-tumor cells);
2. 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 that
identifies epithelial cells as normal or tumor cells is desirable. In that
case,
microscopy based analyses may prove to be the most accurate. Such
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examination might also include examination of morphology, identification of
known tumor diathesis associated molecules (e.g., oncogenes).
Patients
Patients' age range was 47-91 year (mean 74), with initial diagnosis 2 to
years prior to study. Medical records were reviewed for therapy and stage.
Patients and healthy volunteers signed an informed consent under an
approved research study. Blood was drawn into 10mi EDTA VacutainerTM
tubes (Becton-Dickinson, NJ). Samples were kept at room temperature and
10 processed within 6 hours after collection unless indicated otherwise.
Sample Preparation
Magnetic nanoparticles labeled with monoclonal antibodies identifying
epithelial cell adhesion molecule (EpCAM) were used to label and separate by
magnetic means epithelial cells from hematopoietic cells, as taught in
commonly-owned US Patent #6,365,362, and US Patent Application
10/079,939, filed 19 February 2002, both of which are fully incorporated by
reference herein. The magnetically captured cells resuspended in a volume
of 200111 are fluorescently labeled to differentiate between hematopoietic and
epithelial cells. A monoclonal antibody that recognizes keratins 4, 5, 6, 8,
10,
13, and 18, conjugated to Phycoerythrin (CK-PE) was used to identify
epithelial cells and a monoclonal antibody that recognizes CD45 was used to
identify leukocytes and identify hematopoietic cells that non-specifically
bind
to cytokeratin.
For multicolor fluorescent microscopy analysis, CD45 was conjugated to
Allophycocyanin (CD45-APC, Caltag, CA) whereas for flow cytometric
analysis peridinin chlorophyll protein conjugated CD45 (CD45-PerCP, BDIS
San Jose, CA) was used. The nucleic acid specific dye DAPI (4,6-diamidino-
2-phenylindole) was used to identify and visualize the nucleus and the nucleic
acid dye in the Procount system (BDIS, San Jose,CA) was used to identify
cells by flow cytometry. After incubation, the excess staining reagents were
aspirated and the captured cells were resuspended and transferred into a
12x75 mm tube for flow cytometric analysis or image cytoometry anafysis (as
described in US Application 10/074,900, filed 12 February 2002, incorporated
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by reference herein) contained within a magnetic yoke assembly that holds
the chamber between two magnets (Captivate, Molecular Probes, OR).
Example 1
Sample Analysis via Flow Cytometry
Samples were analyzed on a FACSCalibur flow cytometer equipped with a
488nm Argon ion laser (BDIS, San Jose, CA). Data acquisition was performed
with CeIlQuest (BDIS, San Jose, CA) using a threshold on the fluorescence of
the nucleic acid dye. The acquisition was halted after 8000 beads or 80% of
the sample was analyzed. Multiparameter data analysis was performed on
the listmode data (Paint-A-GatePPO, BDIS, San Jose, CA). Analysis criteria for
CTC events included size defined by forward light scatter, granularity defined
by orthogonal light scatter, positive staining with the PE-labeled anti-
cytokeratin MAb and no staining with the PerCP-labeled anti-CD45 Mab. For
each sample, the number of events present in the region typical for epithelial
cells was multiplied by 1.25 to account for the sample volume not analyzed by
flow cytometry.
Figure 2 Panels A, B and C shows flow cytometric analysis of a blood
sample of a patient with metastatic prostate cancer. Two vertical lines in
Panel B illustrate the low and high boundary of nucleic acid (NAD) content of
leukocytes (red dots). CTC candidates express Cytokeratin (CK+), lack CD45
(CD45-) and contain nucleic acids (NAD+). CTC candidates having NAD
equal or higher than leukocytes are considered cells and are depicted black.
CK+, CD45- events with NAD content less than leukocytes were not
considered target cells and depicted blue. The blue events were clearly
smaller as compared with the black colored CTC as evident by the smaller
forward light scatter signals. The threshold on the NAD staining intensity
clearly excluded a large portion of CK+, CD45- events with even lower NAD
staining intensity. In analysis of blood samples from healthy donors few such
CK+, CD45- events are observed suggesting that this phenomenon is related
to cancer. A typical example of an analysis of a blood from a healthy donor is
shown in Figures 2D, 2E, and 2F.
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Example 2
Sample Analysis by Image Cytometry
The image cytometry system consists of a microscope with a Mercury Arc
Lamp, a 10X objective, a high resolution X, Y, Z stage and a four-filter cube
changer. Excitation, dichroic and emission filters in each of four cubes were
for DAPI 365nm/400nm/400nm, for DiOC16 480nm/ 495nm/ 510nm, for PE
546nm/ 560nm/ 580nm and for APC 620nm/ 660nm/ 700nm. Images were
acquired with a digital camera connected to a digital frame grabber. The
surface of the chamber is 80.2 mm2 and 4 rows of 35 images for each of the 4
filters resulting in 560 images have to be acquired to cover the complete
surface. The acquisition program automatically determines the region over
which the images are to be acquired, the number of images to acquire, the
position of each image and the microscope focus to use at each position. All
the images from a sample are logged into a directory that is unique to the
specific sample identification. An algorithm is applied on all of the images
acquired from a sample to search for locations that stain for DAPI and CK-PE.
If the staining area is consistent with that of a potential tumor cell (DAPI+,
CK-
PE+) the software stores the location of these areas in a database. The
software displays thumbnails of each of the boxes and the user can confirm
that the images represented in the row are consistent with tumor cells, or
stain
with the leukocyte marker CD45. The software tabulates the checked boxes
for each sample and the information is stored in the database.
Figure 3 shows examples of image analysis of a blood sample from a
patient with metastatic prostate cancer. Regions that potentially contain
tumor cells are displayed in rows of thumbnails. The ruler in the left lower
corner of the figure indicates the sizes of the thumbnails. From right to left
these thumbnails represent nuclear (DAPI), cytoplasmic cytokeratin (CK-PE),
control cells stained with a membrane dye (DiOC16(3)) and surface CD45
(CD45-APC) staining. The composite images shown at the left show a false
color overlay of the purple nuclear (DAPI) and green cytoplasmic (CK-PE)
staining. The check box beside the composite image allow the user to
confirm that the images represented in the row are consistent with tumor cells
and the check box beside the CD45-APC image is to confirm that a leukocyte
or tumor cell stain non-specifically. In this patient sample, the software
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detected 2761 rows of thumbnails that demonstrated staining consistent with
tumor cells. Eighteen of the 2761 rows are shown in the figure labeled 1631-
1640 and 1869-1876. Rows numbered 1631, 1636, 1638, 1640, and 1873-
1876 are checked off and display features of CTC defined as a size greater
than 40m, the presence of a nucleus surrounded by cytoplasmic cytokeratin
staining and absence of DiOC16(3) and CD45 staining. Note the difference in
appearance of the tumor cells: the cell in row 1638 is large and the one in
row
1640 is significantly smaller. The immunophenotype of the events in rows
1634 and 1869 are consistent with tumor cells but their morphology is not
consistent with intact cells. The thumbnails in row 1869 shows a large
nucleus and speckled cytoplasmic due to retraction of cytoskeletal proteins
consistent with apoptosis of the cell. The thumbnail in row 1634 shows a
damaged cell that appears to extrude its nucleus. The thumbnail shown in
row 1632 shows a cell that stains both with cytokeratin as well as CD45 and is
either a tumor cell non-specifically binding to CD45 or a leukocyte non
specifically staining with cytokeratin. In this instance the morphology of the
cell closely resembles that of a lymphocyte. The thumbnails shown in rows
1633, 1635, 1637, 1639, 1870 and 1872 shows cytokeratin staining objects
that are larger that 4~m but have no resemblance to cells. The cytokeratin
staining objects in thumbnails 1637, 1639 and 1872 are in close proximity of a
leukocyte.
Based on observation of images of CTC candidates in several patient
samples, CTC were classified into three categories: intact CTC, damaged
CTC, and CTC fragments all not staining with CD45 and not appearing in the
DiOC16(3) filter. Figure 4 displays examples of the three categories of CTC
isolated from a single tube of blood of a patient with metastatic prostate
cancer undergoing therapy. Intact tumor cells shown in Figure 3A were
defined as objects larger than 4mm with a relatively smooth cytoplasmic
membrane, cytoskeletal proteins throughout the cytoplasm, and an intact
nucleus encompassed within the nucleus. Damaged CTC shown in Figure 4B
were defined as objects larger than 4mm with speckled cytokeratin staining or
ragged cytoplasmic membrane, and a nucleus associated with the cytokeratin
staining. Tumor cell fragments shown in Figure 4C were defined as round
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cytokeratin staining objects larger than 4mm with or without association of
nuclear material that had no morphological resemblance to a cell.
Example 3
CTC in Prostate Cancer Patients
CTC were enumerated in 18 blood samples of prostate cancer patients
and 27 samples from healthy individuals by both flow cytometry and image
cytometry The results shown in Table I were sorted by increasing number of
intact CTC detected.
Table 1- Enumeration of CTC by image cytometry and flow cytometry in 18
blood samples of prostate cancer patients and 27 samples from healthy
individuals.
Flow
Image C tometr C try
Patient Intact CTC Suspect CTC Not Assigned CK+CD45-
Sample Events NA+
1 0 0 1 50 1 50 5
2 0 0 2 100 0 0 12
3 0 0 2 66 1 34 1
4 0 0 2 50 2 50 0
5 0 0 2 29 5 71 5
6 0 0 3 60 2 40 18
7 0 0 3 38 5 62 0
8 0 0 7 44 9 56 10
9 0 0 13 76 4 24 2
10 1 5 1 5 20 90 4
11 1 10 4 40 5 50 0
12 2 22 1 11 6 67 4
13 28 6 7 1 441 93 69
14 70 5 168 12 1204 83 683
322 3 448 13 4244 87 500
16 350 5 112 2 5924 93 723
17 350 2 1429 9 14412 89 2420
18 742 17 112 2 3641 81 310
Mean - 4% - 34% - 62% -
27 samples from healthy donors
Mean 0.04 0.96 4.96 0.7
SD 0.19 1.85 3.98 1.14
Min 0 0 0 0
Max 1 7 15 4
#- number CTC in 7.5 ml blood % - percentage of all CTC detected by
Image Cytometry
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The proportion of intact CTC clearly constituted the smallest fraction of
CTC and ranged from 0% to 22% of all CTC (mean 4%). The proportion of
damaged CTC ranged from 1% to 100% (mean 34%) and the CTC fragments
constituted the largest portion of CTC ranging from 0% to 93% (mean 62%).
The distribution of CTC over the three categories between the patients varied
considerably as amplified by a lack of correlation between intact CTC and
damaged CTC (R2 = 0.20) and intact CTC and CTC fragments (R2 = 0.42)
and some correlation between damaged CTC and CTC fragments (R2 = 0.88).
Comparison of intact CTC by and CTC enumerated by flow cytometry showed
no significant correlation (R2 = 0.26) whereas significant correlations were
found between the damaged CTC and CTC by flow cytometry (R2 = 0.92) and
CTC fragments and CTC by flow cytometry (R2 = 0.93). Comparison of the
CTC detected by flow cytometry and image cytometry suggests that CTC
detected by flow cytometry encompass intact CTC as well as damaged CTC
and to a certain extent, CTC fragments.
Example 4
Mimicking cell damage by in-vitro induction of apoptosis in LnCaP
cells
,20 To investigate the effect of apoptosis induced by cytotoxic agents on flow
cytometric and image cytometry on CTC, cells from the prostate cell line
LnCaP were cultured in the presence or absence of 40nM paclitaxel for 72
hours. Following incubation, untreated LnCaP cells demonstrated a viability
of >95% by trypan blue exclusion and 33% for the paclitaxel treated cells.
The treated and untreated LnCaP cells were spiked into blood of heaithy
donors, selected by the ferrofluid methods described above, and analyzed by
the image cytometry. In experiments in which LnCaP cells were spiked into
blood that were not treated with paclitaxel greater than 95% of the LnCaP
cells were classified as intact tumor cells. The morphologic appearance of the
paclitaxel treated LnCaP cells showed close resemblance to that of the CTC
observed in the patient samples and are shown in Figure 6. Intact LnCaP
cells that survived paclitaxel treatment are shown in Figure 6A, damaged
LnCaP, of which the majority show speckled cytokeratin staining, are shown
in Figure 6B, and tumor fragments are shown in Figure 6C.
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Normal blood samples spiked with paclitaxel treated and untreated LnCaP
cells were also prepared for flow cytometric analysis. In Figures 2G, 2H, and
21, the flow cytometric analysis of a blood sample spiked with 501 LnCaP cells
is shown. A predominantly bright cytokeratin positive population with a
nucleic acid content greater than normal human leukocytes and relatively
large size as illustrated by the large forward light scatter signals are shown
and depicted black in the figure. Only few CK+, CD45- events with NAD
content less than leukocytes and depicted blue are detected in the sample.
Figures 2J, 2K, and 2L shows the flow cytometric analysis of paclitaxel
treated
LnCaP cells spiked in blood. In contrast to viable LnCaP cells, a wide
distribution of cytokeratin staining was observed with a significant portion
of
the population demonstrating a decreased concentration of nucleic acid
content. In addition, numerous small cytokeratin positive events with less
nucleic acid content as leukocytes were observed. The pattern of the patient
closely resembled that of the pattern of the paclitaxel treated LnCaP cells
supporting the hypothesis that the CTC detected by flow cytometry represent
intact CTC as well as a variety of disintegrating cells in blood of cancer
patients.
The data shown above demonstrate that in the blood of patients with
prostate cancer, CTC detected by both flow cytometry and image cytometry
are comprised of intact cells and cells of cells at various stages of
disintegration. The apoptosis induced in vitro by paclitaxel suggests that the
detected CTC in patient blood samples are undergoing apoptosis, necrosis, or
in vivo damage to a varying degree caused by the treatment or therapy,
mechanical damage by passage through the vascular system, or by the
immune system.
Another source of cell disintegration, caused in vitro could however, be
introduced by the sample preparation or the lack of stabilization of CTC or
other blood components after blood draw. To investigate the effect of sample
aging, known to cause damage, blood samples drawn from 12 patients with
prostate cancer were processed and analyzed by flow cytometry within two
hours, after 24 hours, and after 6 and 18 hours if sufficient blood was
available. In 8 of the 12 patient samples, CTC were detected at a level
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greater than the mean +3SD of that detected in normal donors. As shown in
Table 2, a loss of CTC with sample aging was observed in all 8 samples.
Table 2 - Enumeration of CTC by flow cytometry in 8 blood samples of
prostate cancer patients processed and analyzed at different time points after
blood draw
Time after blood draw
Patient # < 2hr -6hr -18hr -24hr
#CTC #CTC #CTC #CTC
1 5 - - 0
2 8 9 2 3
3 15 - - 0
4 31 - - 3
5 44 - - 8
6 45 - - 1
7 49 38 19 26
8 78 - - 0
hr = hours #CTC = number of CTC in 5 mi blood
Significant reductions in the number of CTC were detected when blood
processing was delayed demonstrating the fragility of CTC, and making it
necessary to process non-stabilized blood samples no later than six hours
after blood draw to obtain accurate CTC counts. To reliably assess if
clinically
relevant information is contained within the different stages of tumor cell
degradation, a blood preservative is needed that stabilizes CTC at the time of
blood draw to obtain an accurate reflection of what is occurring inside the
body. Furthermore, the sample preparation method for sensitive assays used
to enrich for CTC requires that all classes of CTC are captured, and therefore
excludes the use of traditional density gradient separation methods in the
prior art.
Example 5
Obvious CTC and Suspect CTC are important indicators
It is important to be able to distinguish between in vivo and in vitro damage
for sensitive assays, such as those described here. This is especially evident
when the assay attempts to determine the effectiveness of treatments or
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therapies, which are known to cause in vivo cellular damage. If sample
handling, processing, or analysis were to result in damaging the target cells,
forming Suspect cells, fragments, or debris, the assay will not give
meaningful
results.
An assay was used to directly detect CTC in 100111 of blood without any
enrichment method by flow cytometry. The 100~I assay detects only EpCAM
positive cells and the sensitivity is very low. However, some advanced stage
cancer patients with high CTC counts are expected to be observable. This
assay should give a reliable confirmatory estimation of CTC because it is a
direct assay that involves no manipulation. Data were generated with several
patient samples using the assay to answer several questions.
The 10001 assay categorizes cells based on properties such as size and
staining intensity. Obvious CTC have bright nucleic acid staining (similar to
leukocytes), positive EpCAM antigen staining and size similar to leukocytes or
larger. Suspect CTC are any objects positive for EpCAM but not
characterized as Obvious CTC (i.e. dim nucleic acid, size smaller than
leukocytes). The assay identifies objects from both categories.
Figure 5 shows the presence of obvious and suspect CTC in blood as
determined by the 10001 assay. The Suspect CTC are not created during
sample processing (in vitro damage) as the 10001 assay is a direct assay and
does not involve any separation or wash steps. The data above also show
there is a relationship between the number of Obvious and Suspect CTC.
The number of Suspect CTC seems to increase as the number of Obvious
CTC increases. When the numbers of Suspect versus Obvious CTC is
plotted, the slope of 2.92 indicates the proportion of Suspect CTC present in
sample when compared to Obvious CTC. The correlation coefficient of r2 =
0Ø97 shows an excellent correlation between Obvious CTC and Suspect
CTC for a number of clinical samples. In addition, Suspect CTC are also
seen in the ferrofluid-selection assay, and have properties similar to Suspect
CTC detected in the blood by the direct assay. It is important to include
Suspect CTC in addition to Obvious CTC in total tumor cell count.
An important question is how the data from the 100~I assay compares
with ferrofluid-selected CTC (enriched CTC). Does the ferrofluid assay
quantitatively detect CTC? Another question is what is the recovery of CTC in
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the ferrofluid-selected assay if the flow assay data is correct. The three
main
factors determining the recovery of CTC in the assay are:
= EpCAM density,
= cytokeratin positivity, and
= nucleus positivity.
The suspect CTC have lower EpCAM density compared to obvious CTC
and significance of this is not yet well understood.
A comparison was made of obvious and suspect CTC by the 100 ml
assay to the ferrofluid-selection assay using 7.5 ml of blood. This data was
obtained from prostate patient samples and analyzed by flow cytometry. Both
obvious and suspect CTC increased with storage time and the trend was
similar to CTC detected in the ferrofluid-selection assay, thereby validating
the
100~I assay. The recovery of CTC from the ferrofluid-selection assay was
about 90% based on the CTC in 100 ml of blood. It was also known that MFI
(Mean Fluorescence Intensity which correlates the EpCAM density) of CTC
from this patient was high (MFI=300), and all EpCAM positive cells are
cytokeratin positive. However, the recoveries of CTC from some other clinical
samples have been as, low as 20%. There may be several factors that
contribute for a lower recovery, such as EpCAM positive/cytokeratin negative
cells, cytokeratin dim cells, and mucin on the cell surface inhibiting the
ability
of ferrofluid to bind cells.
The assay described herein was performed on patients at two times.
Response was measured by bi-dimensional imaging of the lesion. The Ratio
(Ratio = Obvious CTC / Total CTC) is similar to the Response Index described
earlier, and can be used as a numeric indicator of treatment success. The
results are summarized in Table Ill. Ratios near 1.0 indicate the Total CTC
are obvious CTC, and ratios near 0.0 indicate more suspect CTC or debris.
Progressive indicates the lesion increasing in size, partial response
indicates
a response to treatment where the Ratio is relatively low, and Stabilized
indicates no change, or reduction in lesion size. A positive change indicates
an increase in the number of Intact CTC, corresponding to the progression of
the disease. A negative change indicates a decrease in the number of Intact
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CTC, or a possible increase in the number of suspect CTC and/or debris,
corresponding to a response to treatment.
These results show the importance of including suspect CTC and debris
when analyzing response to treatment because the numbers of intact or
obvious CTC alone would not provide as much information. Furthermore,
such indicators are useful for short-term monitoring of treatments and
therapies, or longer term monitoring for remission and/or relapse.
Table 3- Obvious CTC and Suspect CTC corresponding to treatment
response
Response Ratiol Ratio2 Change
0.3 0.0 -0.3
4) 0.0 0.0 0.0
> 0.5 0.6 0.1
N 0.9 1.0 0.1
L 0.3 0.5 0.2
0.4 0.7 - 0.3
O
0.0 0.4 0.4
a
0.5 0.9 0.4
0.0 0.5 0.5
0.0 0.6 0.6
1.0 0.0 -1.0
~ N 0.4 0.0 -0.4
C 0.3 0.0 -0.3
m 0.5 0.2 -0.3
0' N 0.4 0.3 -0.1
0.0 0.0 0.0
0.3 1.0 0.7
1.0 0.0 -1.0
0.5 0.0 -0.5
1.0 0.7 -0.3
.a 0.3 0.0 -0.3
N 0.4 0.3 -0.1
0.1 0.0 -0.1
B 0.2 0.1 -0.1
0.6 0.5 -0.1
0.9 0.8 -0.1
0.0 0.0 0.0
0.6 0.7 0.1
0.6 0.8 0.2
0.0 1.0 1.0
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Example 6
Reduction in mRNA content after plasma washing.
Patients with >20 CTC/7.5 ml of blood were assayed for the quantity of
CTC specific mRNA. Individual samples from patients (n=12) were matched
by dividing a patient's sample into plasma wash and no plasma wash groups
after addition of cell preservative (U.S. Appl. No. 10/780,349). Following
EpCAM magnetic enrichment and centrifugation, PSA mRNA remaining in the
enriched fraction was assessed by quantitative RT-PCR. The table below
shows that plasma washings, either at the time of blood draw (0 hr EDTA) or
24 hr after blood draw, resulted in a significant loss of mRNA.
Table 4- Reduced CTC specific mRNA for PSA after plasma wash
Q RT-PCR PSA Average Median Range Wilcoxon
X3' Assay & Fold Fold Signed-
Method Difference Difference Rank
Comparison
24 hr 3.1 2.4 0.3 to 9 (p = 0.05)
(No
Wash/Wash)
0 hr EDTA 11 7 2 to 33 (p = 0.005)
(No Wash/24 hr
No Wash)
O hr EDTA 46 14 1 to 187 (p = 0.006)
(No Wash/24 hr
Wash)
Table 4 shows that plasma washing eliminates at least 3 fold mRNA.
Because intact CTCs do not remain in the plasma following centrifugation at
800(g), RNA signals must come from a fraction of cell debris that does not
partition from the plasma fraction and remains in the plasma, subsequently
aspirated away with washing. Consequently, an even larger difference could
result with the incorporation of rare cell debris, partitioned from the plasma
after centrifugation.
Example 7
The presence of gene expression in enriched blood samples of
patients with metastatic colorectal cancer devoid of intact CTC.
Two 7.5 ml blood samples from 37 healthy donors and 49 patients with
CRC were immunomagnetically enriched by targeting the EpCAM antigen.
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One sample was used to determine the number of intact CD45-, cytokeratin 8,
18 and/or 19 positive tumor cells and the other sample was used to evaluate
the expression of CK20, CEA, CK19, EGFR, GUC, EpCAM, VEGF, TS and
Muc-1. Gene expression was evaluated by creating an aRNA library from the
CTC enriched samples followed by RT-PCR of the individual genes.
No intact CTC and no RT-PCR signals for CK19, CK20, CEA, and EGFR
were detected in the control group. RT-PCR for GUC, EpCAM, VEGF, TS
and Muc1 showed positive signals in the control group and were not further
evaluated in CRC. In 19 of 49 (39%) samples of CRC patients, 2 or more
CTC were detected in 7.5 ml blood samples. The number and percentage of
samples that scored positive for CK20, CEA, CK19, EGFR in the samples
with and without intact CTC are listed in the table 5 below.
Table 5- Comparison of RT-PCR signals from enriched samples with/without
intact CTC.
CK20 CEA CKI 9 EGFR Genes
Combined
CTC Negative 9 16 17 1 18
(n = 30) (30%) (53%) (57%) (3%) (60%)
CTC Positive 14 14 14 4 15
n= 19 (74% 74%) (74%) 21 % 79%)
Total n= 49 23 30 31 5 33
(47%) 61 % (63%) (10%) (67%)
In 15 of 19 (79%) CRC samples with intact CTC one of the genes was
expressed but surprisingly in 18 of 30 (60%) samples with no intact CTC
scored positive for at least one of the genes from the gene panel.
Intact CTC and genes expressed in epithelial cells can be detected in
blood samples of CRC patients enriched for EpCAM expression. The finding
of RT-PCR positives in patients in which no intact CTC were detected may be
due to carcinoma cells shed into the blood that have been damaged or
destroyed.
Enumeration of tumor cell debris may prove more significant in cancer
diagnostics and therapeutics than detection of large proliferative cell
clusters.
Since debris particles in the size range, probably about 1-30m (the size of
platelets), have been observed to be present in much larger amounts than
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intact cells, they may constitute a separate, independent, and possibly more
sensitive marker than intact tumor cells. The presence of damaged CTC may
be particularly relevant in detecting early-stage cancer, when the immune
system is intact and most active. Similarly, dramatic increases in debris
during therapy may suggest breakdown of both circulating and tissue tumor
cells (i.e. therapeutic effectiveness), paralleling the massive release of
cellular
components like calcium observed during tumor disintegration. Like soluble
tumor markers, such debris may be detectable in blood without enrichment, or
with minimal enrichment in the buffy coat layer and constitute an alternative,
and potentially simpler diagnostic tool than intact cell enrichment/analysis.
Since morphology is lost in CTC debris, detection could be done by flow
cytometry as long as the debris is stained for the appropriate determinants,
such as cytokeratin.
As previously discussed, damaged or fragmented CTC with or without
DNA are theoretically to be expected, and therefore are not undesirable
events in specimens from patients undergoing effective therapy and in
untreated patients with strong immune systems. The ratio or percent of intact
CTC to total detectable events may prove to be a more useful parameter to
the clinician in assessing a patient's immune system or response to therapy.
The normal immune defenses, especially activated neutrophils, also can
damage or destroy CTC as foreign species by a process called "extracellular
killing" even if the CTC are larger than the neutrophils. It does not seem
surprising to find only a small percentage of the shed CTC as intact cells,
unless the immune system is overwhelmed in the late stages of disease or
therapy is ineffective.
Hence, there are a number of methods for in vitro cancer detection:
conclusive detection of intact circulating cells/clusters, and inferential
methods
like circulating tumor debris (including total and tumor-specific RNA/DNA, and
conventional soluble tumor markers. However, no method by itself may be
sufficiently sensitive. Lower specificity of debris detection compared to CTC
morphology may be a problem in screening that could be minimized (e.g. with
triple labeling), but it may be a lesser problem in monitoring. Further
statistical analysis and correlations on debris data relative to intact CTC
and
diagnostic stage in patients compared to normals appear worthwhile in
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assessing the sensitivity and specificity of debris analysis. This data would
include sequence analysis of nucleic acid or protein components from rare
cells in the enriched samples. Sequence analysis includes the quantification,
and/or qualification of an individual sequence or groups of sequences
associated with the disease of interest. For example, RNA sequence analysis
is accomplished by multigene RNA profile analysis. Sequence quantification
is accomplished by quantitative RT-PCR while sequence qualification is
accomplished through array analysis. The present invention is not limited to
this analysis, but includes all sequence analysis accepted by individuals in
the
field.
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,
Ieiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma,
rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma,
medulloblastoma, meningioma, neurilemmoma, neuroblastoma,
neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma
nonchromaffin, antiokeratoma, angioma sclerosing, angiomatosis,
glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma,
hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma,
pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes,
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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.
However, the present invention is not limited to the detection of circulating
epithelial cells and/or clusters, fragments, or debris. For example,
endothelial
cells have been observed in the blood of patients having a myocardial
infarction. Endothelial cells, myocardial cells, and virally infected cells,
like
epithelial cells, have cell type specific determinants that are 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. It would
be reasonable to expect that these rare cells will behave similarly in
circulation, and that fragments and/or debris will be present in similar
conditions as those described hereinabove.
The preferred embodiments of the invention as herein disclosed, are also
believed to enable the invention to be employed in fields and applications
additional to cancer diagnosis. It will be apparent to those skilled in the
art
that the improved diagnostic modes of the invention are not to be limited by
the foregoing descriptions of preferred embodiments. Finally, while certain
embodiments presented above provide detailed descriptions, the following
claims are not limited in scope by the detailed descriptions. Indeed, various
modifications may be made thereto without departing from the spirit of the
following claims.
While the preferred embodiments of the present invention as disclosed
relate to cancer and cardiovascular diagnostics, those skilled in the art will
recognize that the invention is enabling in other fields and applications.
Thus,
it is apparent that the improved diagnostic modes of the present invention are
not to be limited by the foregoing descriptions. While certain embodiments
presented above provide detailed descriptions, the following claims are not
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limited in scope by the detailed descriptions. Accordingly, various
modifications may be made thereto without departing from the spirit of the
following claims.
47
