Method of Using Non-Rare Cells to Detect Rare Cells

Abstract

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.

1 . A method for detecting a cell in a sample from a subject comprising:
 a) 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;   b) contacting the sample with at least one detectable agent;   c) performing cell imaging on the sample of (b) to generate a cell image; and   d) detecting the at least one rare cell as compared with other cells in the sample by analyzing the cell from the image of (c), thereby detecting the rare cell in the sample.   
     
     
         2 . The method of  claim 1 , further comprising (a′), wherein the at least one rare cell and at least one non-rare cell are affixed to a slide. 
     
     
         3 . The method of  claim 1 , wherein (d) is performed on a computer comprising executable code for performing the analysis. 
     
     
         4 . The method of  claim 3 , further comprising (e), wherein the results of (d) are presented on a graphical user interface. 
     
     
         5 . The method of  claim 1 , wherein the cell imaging of (c) further comprises optimizing an exposure limit for the at least one detectable agent. 
     
     
         6 . The method of  claim 5 , wherein the exposure limit for the at least one detectable agent is determined using a signal from the at least one non-rare. 
     
     
         7 . The method of  claim 5 , wherein the at least one detectable agent binds a cell marker and is selected from a positive marker, negative marker, nuclear marker, content marker, and any combination thereof. 
     
     
         8 . The method of  claim 5 , wherein the exposure limit is optimized before generating the image. 
     
     
         9 . The method of  claim 5 , wherein the exposure limit is optimized after generating the image. 
     
     
         10 . The method of  claim 5 , further comprising (c′), wherein the sample is re-imaged prior to performing (d). 
     
     
         11 . The method of  claim 1 , wherein the cell imaging of (c) further comprises optimizing the exposure time to decrease data collection time. 
     
     
         12 . The method of  claim 1 , wherein a signal from the at least one cell is used to determine a quality control parameter. 
     
     
         13 . The method of  claim 12 , wherein the quality control parameter is distribution of the at least one cell on the slide. 
     
     
         14 . The method of  claim 12 , wherein the quality control parameter is alignment of multiple cell images. 
     
     
         15 . The method of  claim 14 , wherein the at least one detectable agent is a negative marker and a nuclear marker and the alignment is determined by aligning a signal from the nuclear marker with a signal from the negative marker, each present on different images. 
     
     
         16 . The method of  claim 12 , wherein the quality control parameter is quality of cell staining. 
     
     
         17 . The method of  claim 16 , wherein the at least one detectable agent is a negative marker and a nuclear marker and the quality of cell staining is determined by calculating a ratio of signals from the negative markers to signals from the nuclear markers. 
     
     
         18 . The method of  claim 17 , wherein the ratio is less than 1.2. 
     
     
         19 . The method of  claim 12 , wherein the at least one detectable agent is a negative marker and the quality control parameter is distribution of the negative marker throughout the non-rare cells. 
     
     
         20 . The method of  claim 19 , wherein the distribution is calculated using the mean, standard deviation, coefficient of variation, or any combination thereof. 
     
     
         21 . The method of  claim 12 , wherein the at least one detectable agent is a positive marker and the quality control parameter is distribution of the positive marker throughout the non-rare cells. 
     
     
         22 . The method of  claim 21 , wherein the distribution is calculated using the mean, standard deviation, coefficient of variation, or any combination thereof. 
     
     
         23 . The method of  claim 12 , wherein the quality control parameter is cell loss. 
     
     
         24 . The method of  claim 23 , wherein the at least one detectable agent is a nuclear marker or a negative marker and cell loss is determined by calculating a ratio of signals from the nuclear markers or signals from the negative markers to the number of non-rare cells on the slide. 
     
     
         25 . The method of  claim 1 , wherein the cell imaging of (c) further comprises optimizing intensity limits to distinguish rare cells from other cells. 
     
     
         26 . The method of  claim 25 , wherein the at least one detectable agent is a positive marker and the intensity limits are determined using mean, standard deviation, coefficient of variation, or any combination thereof, for a background signal of the positive marker. 
     
     
         27 . The method of  claim 26 , wherein the mean, standard deviation, or coefficient of variation are determined on a global slide basis, single slide basis, region of a single slide, or any combination thereof. 
     
     
         28 . The method of  claim 25 , wherein the at least one detectable agent is a positive marker and the intensity limits are determined within a single image by identifying a highest signal of the positive marker and comparing the highest signal to a standard deviation calculated from signals of additional positive marker. 
     
     
         29 . The method of  claim 26 , wherein the intensity limit is equal to a factor of between 5 and 20 multiplied by the standard deviation and added to the mean. 
     
     
         30 . The method of  claim 29 , wherein the intensity limit is equal to a factor of 12.5 multiplied by the standard deviation and added to the mean. 
     
     
         31 . The method of  claim 30 , wherein the intensity limit is equal to a factor of 15 multiplied by the standard deviation and added to the mean. 
     
     
         32 . The method of  claim 29 , wherein the intensity limit is calculated for each sample image. 
     
     
         33 . The method of  claim 25 , wherein the at least one 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. 
     
     
         34 . The method of  claim 33 , wherein the mean and standard deviation are determined on a global slide basis, single slide basis, region of a single slide, or any combination thereof. 
     
     
         35 . The method of  claim 1 , wherein (d) further comprises measuring cellular sizes for the at least one cell, distribution of cellular sizes for the at least on cell, or combination thereof, and comparing the cellular sizes or distribution of cellular sizes for the at least one cell, to known cellular sizes and distributions. 
     
     
         36 . The method of  claim 1 , wherein the analysis of (d) further comprises measuring nuclear sizes for the at least one cell, distribution of nuclear sizes for the at least one cell, contour patterns for the at least one cell, or combination thereof, and comparing the nuclear sizes, distribution of nuclear sizes or contour patterns to a putative rare cell to identify the suspected rare cell as a rare cell. 
     
     
         37 . The method of  claim 1 , wherein the analysis of (d) further comprises determining the concentration of rare cells in a fluid of the subject from which the sample was taken. 
     
     
         38 . The method of  claim 1 , wherein the detectable agent is a content marker and the analysis of (d) further comprises determining an expression level of the content marker. 
     
     
         39 . The method of  claim 36 , wherein the expression level of the content marker is determined in the at least one rare cell and in the at least one cell and compared to the expression level of the content marker in a patient population. 
     
     
         40 . The method of  claim 1 , wherein the rare cell is a circulating tumor cell (CTC). 
     
     
         41 . The method of  claim 40 , wherein the CTC expresses a positive marker, has an intact nucleus, and is morphologically distinct from a normal WBCs, wherein the CTC is not positive for a negative marker. 
     
     
         42 . The method of  claim 41 , wherein the positive marker is cytokeratin or EpCAM. 
     
     
         43 . The method of  claim 41 , wherein the negative marker is CD45. 
     
     
         44 . The method of  claim 40 , further comprising providing a diagnosis or prognosis to the subject. 
     
     
         45 . The method of  claim 40 , wherein the subject is known to have cancer and is undergoing cancer therapy. 
     
     
         46 . The method of  claim 45 , wherein the therapy is chemotherapy. 
     
     
         47 . The method of  claim 46 , wherein a content marker is used to determine a chemotherapeutic agent. 
     
     
         48 . The method of  claim 44 , wherein the subject is being administered a candidate agent. 
     
     
         49 . The method of  claim 44 , further comprising determining the responsiveness of the subject to the cancer therapy. 
     
     
         50 . A method for diagnosing or prognosing cancer in a subject comprising:
 a) performing the method of  claim 40 ; and   b) analyzing the detected CTC to provide a diagnosis or prognosis, thereby diagnosing or prognosing cancer in a subject.   
     
     
         51 . A method for determining responsiveness of a subject to a therapeutic regime comprising:
 a) performing the method of  claim 40 ; and   b) analyzing the detected CTC, thereby determining the responsiveness of the subject to a therapeutic regime.   
     
     
         52 . The method of  claim 51 , wherein the therapeutic regime is changed based on the results of b). 
     
     
         53 . A method for determining a candidate subject for a clinical trial comprising:
 a) performing the method of  claim 40 ; and   b) analyzing the detected CTC, thereby determining a candidate subject for a clinical trial.