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Method of using non-rare cells to detect rare cells

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Method of using non-rare cells to detect rare cells

The invention provides seminal computational approaches utilizing data from non-rare cells to detect rare cells, such as circulating tumor cells (CTCs). The invention is applicable at two distinct stages of CTC detection; the first being to make decisions about data collection parameters and the second being to make decisions during data reduction and analysis. Additionally, the invention utilizes both one and multi-dimensional parameterized data in a decision making process.
Related Terms: Circulating Tumor Cells

Inventors: Peter Kuhn, Anand Kolatkar, Joshua Kunken, Dena Marrinucci, Xing Yang, John R. Stuelpnagel
USPTO Applicaton #: #20120276555 - Class: 435 723 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate >Animal Cell >Tumor Cell Or Cancer Cell

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The Patent Description & Claims data below is from USPTO Patent Application 20120276555, Method of using non-rare cells to detect rare cells.

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1. Field of the Invention

The invention relates generally to medical diagnostics and more specifically to detection and categorization of rare cells, such as circulating tumor cells (CTCs).

2. Background Information

Significant unmet medical need exists for the longitudinal disease monitoring in patients with epithelial cancers at the cellular level. Predicting and monitoring therapy response and disease progression are particularly important in epithelial cancer patients due to the natural history of the disease and the selective selection process in response to the therapeutic pressure. While progress has been made in understanding the primary and metastatic tumors in their respective microenvironments, a substantial barrier exists in understanding carcinoma behavior during the fluid phase, as it spreads within and occupies the bloodstream. The circulating component of cancer contains within it the cells giving rise to future metastases, and as such, represents a compelling target for investigation.

Research to fully characterize the clinical significance of this fluid phase of solid tumors has been hindered by the lack of easily accessible and reliable experimental tools for the identification of CTCs. The unknown character and low and unknown frequency of CTCs in the blood, combined with the difficulty of distinguishing between cancerous versus normal epithelial cells, has significantly impeded research into how the fluid phase might be clinically important. The ideal fluid phase biopsy should find significant numbers of a specific CTC population in most epithelial cancer patients and preserve and present CTCs to a pathologist and/or researcher in a format that enables not only enumeration but further molecular, morphologic and/or phenotypic analysis. In addition, it should preserve the remaining rare populations for further analysis.

CTCs are generally, although not exclusively, epithelial cells that originate from a solid tumor in very low concentration and enter into the blood stream of patients with various types of cancer. CTCs are also thought to be capable of originating in the blood, forming small colonies throughout the body. The shedding of CTCs by an existing tumor or metastasis often results in formation of secondary tumors. Secondary tumors typically go undetected and lead to 90% of all cancer deaths. Circulating tumor cells provide the link between the primary and metastatic tumors. This leads to the promise of using the identification and characterization of circulating tumor cells for the early detection and treatment management of metastatic epithelial malignancies. Detection of CTCs in cancer patients offers an effective tool in early diagnosis of primary or secondary cancer growth and determining the prognosis of cancer patients undergoing cancer treatment because number and characterization of CTCs present in the blood of such patients has been correlated with overall prognosis and response to therapy. Accordingly, CTCs serve as an early indicator of tumor expansion or metastasis before the appearance of clinical symptoms.

While the detection of CTCs has important prognostic and potential therapeutic implications in the management and treatment of cancer, because of their occult nature in the bloodstream, these rare cells are not easily detected. CTCs were first described in the 1800s, however only recent technological advances have allowed their reliable detection. The challenge in the detection of circulating tumor cells is that they are present in relatively low frequency compared to other nucleated cells, commonly less than 1:100,000. To compensate for this challenge, most conventional approaches for detecting circulating tumor cells rely on experimental enrichment methods, whereby the CTCs are preferentially separated from the other cellular components (e.g., non-CTCs), most importantly other nucleated cells that are the most similar to CTCs.

Currently, the most utilized methods of positive enrichment for enumeration/characterization of CTCs are immunomagnetic enrichment methods targeting the surface protein EpCAM and the “CTC chip”. The most widely used methodology to detect CTCs, J&J\'s Veridex technology, utilizes immunomagnetic enrichment. The technology relies upon immunomagnetic enrichment of tumor cell populations using magnetic ferrofluids linked to an antibody which binds epithelial cell adhesion molecule (EpCAM), expressed only on epithelial derived cells. This methodology requires 7.5 mL of blood for analysis and finds greater than 2 CTCs in only some metastatic cancer patients.

Microfluidic or “CTC-Chip” technology, is another positive enrichment method for enumeration/characterization of CTCs. The methods utilizes 1-3 mL of blood in which whole blood flows past 78,000 EpCAM-coated microposts. EpCAM+ cells stick to the posts and are subsequently stained with cytokeratin, CD45, and DAPI. With this methodology, CTCs are found in virtually all metastatic cancer patients at a relatively high purity and not in healthy controls. Additionally, CTC-chip technology identifies CTCs in all patients and in higher numbers than other technologies by a factor of approximately 10 to 100 fold as reported in two recent publications.

The only routinely used technology for CTC detection is based on immunomagnetic enrichment. This current “gold standard” and FDA approved test is called CellSearch® and employs an immunomagnetic enrichment step to isolate cells that express the epithelial cells adhesion molecule (EpCAM). Additionally, to be identified as a CTC, the cell must contain a nucleus, express cytoplasmic cytokeratin, and have a diameter larger than five microns. This system has uncovered the prognostic utility of enumerating and monitoring CTC counts in patients with metastatic breast, prostate, and colorectal cancers; however, the sensitivity of this system is low, finding no or few CTCs in most patients. Most follow-on CTC technologies have reported higher sensitivity and are pursuing variations of the enrichment strategy, however this directly biases the detectable events towards those that have sufficient expression of the protein selected for the initial enrichment step.

A standardized microscope based approach has also been previously utilized to identify and morphologically characterize and credential CTCs in case studies of breast, colorectal, and lung cancer patients.

Although many CTC detection approaches are currently in use, significant limitations have been identified with the current approaches. For example, one significant limitation of positive selection methods to enumerate/characterize CTCs is that positive physical selection invariably leads to loss of CTCs and is less than 100% efficient. Thus the number of CTCs detected per sample using current methods is often too low to provide robust interpretation or clinically meaningful content of a particular sample. Additional limitations of current methods include low CTC detection due to CTC heterogeneity. For example, differences in individual CTC features within the CTC population of interest further hinder the number of CTCs detected using current methodologies. Such differences may include size variations between individual CTCs, and variable or down regulated expression between individual CTCs of the cell surface markers used to detect CTCs. A further limitation of existing methodologies includes limitations in purity levels and variable purity. Any enrichment will have a certain number of false positives, for instance other nucleated blood cells that stick to the enrichment. For example, the Veridex magnet has typically 5,000 to 10,000 false positives on top of the 5 to 10 positives.



The present invention is based in part on the discovery of innovative methods for analyzing samples to detect, enumerate and characterize rare cells, such as CTCs. Accordingly, the present invention provides methods for improved detection and characterization allowing for clinically meaningful analysis of samples for use in clinical, research and development settings.

Accordingly, the present invention provides methods for the improved detection and characterization of rare cells in a sample by utilizing data from non-rare cells (cells present at a concentration of 10, 50, 100, 200, 300, 400, 500, 1,000, 5,000, 10,000 times or greater as compared to the rare cell) in the sample. Thus the method of the invention utilizes similarity measures to assess non-similarity of cells, requiring both the biggest distance exclusion, e.g., events that are clearly non-rare cell related and the fine distinction of a cutoff based on similarities of surrounding non-rare cells.

The method includes providing a sample suspected of having at least one rare cell and at least one cell that is present at a concentration that is at least 10 times that of the rare cell; contacting the sample with at least one detectable agent, such as an agent that binds a cell marker; performing cell imaging on the sample to generate an image; and detecting the at least one rare cell as compared with other cells in the sample by analyzing the cell from the image, thereby detecting the rare cell in the sample. In various aspects of the invention, the method further includes plating of the suspected rare cell and at least one cell on a solid support, such as a slide, to facilitate contacting the cells with the detectable agent and cell imaging. In various aspects of the invention, the detectable agent is any agent used to stain the cells, such as an agent that binds a cell marker, including, but not limited to, a positive marker, negative marker, nuclear marker, content marker, or any combination thereof.

In various aspects of the invention, the methods described herein are performed on an apparatus for efficiently imaging a slide containing a detectable signal, such as a fluorescent signal. The apparatus may typically include a computer having at least one system processor with image processing capability, a computer monitor, an input device, a power supply and a microscope subsystem. Thus the apparatus includes a computer having executable code for performing the various analysis required to practice the invention. The microscope subsystem includes an optical sensing array for acquiring images. A two-dimensional motion stage for sample movement and for focus adjustment, and input and output mechanisms for multiple sample analysis and storage. The apparatus may also include a transmitted light source as well as an illuminating/fluorescent excitation light source for fluorescing samples.

In one embodiment of the invention, the method includes establishing optimal exposure limits for performing the cell imaging that facilitate detection of rare cells present. In one aspect, the exposure limit for the detectable agent is determined using a signal from at least one cell. In various aspects, the detectable marker may be a positive marker, negative marker, nuclear marker or content marker. In a related aspect, the exposure limits may be set using data relating to the cells and/or suspected rare cells gathered from a first image, to re-image the slide.

In another embodiment, the method includes minimizing exposure settings to minimize data collection time and maximize throughput to facilitate detection of rare cells.

In another embodiment, the method includes utilizing data associated with non-rare cells to generate a quality control parameter that facilitates detection of rare cells. In various aspects, the quality control parameter is distribution of at least one non-rare cell on the slide, alignment of multiple cell images via alignment of non-rare cell markers, quality of cell staining, distribution of a positive marker throughout the non-rare cells, or cell loss from repeated processing.

In another embodiment, the method includes determining intensity cut-off limits to minimize false negatives, as well as false positives and to facilitate rare cell detection. In one aspect, the detectable agent is a positive marker and the intensity limits are determined using mean, standard deviation, coefficient of variation, other statistical parameters or any combination thereof, for a background signal of the positive marker. In another aspect, the detectable agent is a positive marker and the intensity limits are determined within a single image, or portions of that image, by identifying the highest signal event from a positive marker and comparing the highest signal to the mean and standard deviation calculated from signals of all, or a subset of events. In yet another aspect, the detectable agent is a negative marker and the intensity limit for the negative marker is determined using mean and standard deviation of signals from the negative markers from non-rare cells (either all non-rare cells or a specific subset).

In another embodiment, cytological features of non-rare cells, such as cellular and nuclear size (absolute and relative; overall and apparent) and distribution, are utilized to facilitate detection of non-rare cells.

In another embodiment, the method includes utilizing data associated with non-rare cells to enumerate rare cells, thus facilitating their detection. In various embodiments, data may include, but is not limited to, total intensity, mean intensity, segmented intensity, fixed circle, variable circle, or any combination thereof.

In another embodiment, the method includes determination of the expression level of a content marker in rare cells and non-rare cells to facilitate detection of rare cells.

In various aspects of the invention, a rare cell is a CTC or subpopulation thereof.

As such, in another embodiment, the invention provides a method for diagnosing or prognosing cancer in a subject. The method includes performing the method of improved detection and characterization of CTCs as described herein and analyzing detected CTCs and provide a diagnosis or prognosis based on analysis of the CTCs, thereby diagnosing or prognosing cancer in a subject.

In another embodiment, the invention provides a method for determining responsiveness of a subject to a therapeutic regime. The method includes performing the method of improved detection and characterization of CTCs as described herein and analyzing the CTCs, thereby determining the responsiveness of the subject to a therapeutic regime.

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Application #
US 20120276555 A1
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Circulating Tumor Cells

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