Process for multi-analyses of rare cells extracted or isolated from biological samples through filtration

Abstract

A process for isolating or extracting rare cells from a biological sample comprising filtering a biological sample, which may be treated or diluted, through a filter that has a pore size, pore density or other physical properties that retain rare cells, but which permits other kinds of cells to pass through the filter. This process also comprises multiple analyses performed on rare cells after their extraction or isolation by filtration to diagnostically identify the presence of rare cells in a biological sample and to use their diagnostic identification and molecular characterization for diagnostic purposes such as for early diagnosis of diseases, namely for early diagnosis of cancer and to select, guide, monitor treatments and in particular to select targeted treatments and to monitor the response and/or resistance to them. A kit comprising tools, equipment and/or reagents to accomplish both the filtration step and various kinds of multiple analyses performed after isolation and extraction of the rare cells by filtration.

1 . A process for identifying, diagnosing, or providing a prognosis for a condition, disorder or disease associated with rare cells comprising:
 (a) isolating or extracting rare cells by passing a biological sample through a filter and recovering the isolated rare cells on the filter; wherein the filter has a pore size, pore density or other physical characteristics that retain rare cells but which permit passage of other kinds of cells;   (b) determining the cytomorphology of the isolated or extracted rare cells, and/or immunolabeling the isolated rare cells, and/or performing molecular analysis on the isolated rare cells; and   (c) identifying, diagnosing, or providing a prognosis for a condition, disorder or disease and/or a stage of a condition, disorder or disease associated with the rare cells presence and/or number and/or characteristics based on the cytomorphology, and/or immunolabeling, and/or molecular analysis of the isolated or extracted rare cells.   
     
     
         2 . The process of  claim 1 , wherein the biological sample is blood, a biological liquids, mucus, or a sample obtained from a mucus membrane. 
     
     
         3 . The process of  claim 1 , wherein the sample is obtained from a subject having or suspected of having himself or carrying a fetus with a non-cancerous or non-tumorous condition or disorder, including a genetic disease or disorder or a disease selected from the group consisting of an inflammatory, degenerative, cardiovascular, and infectious condition, disorder or disease. 
     
     
         4 . The process of  claim 1 , wherein the biological sample is obtained from a subject who has cancer or a non-cancerous proliferative condition, disorder, or disease, or who is suspected of having or at risk of having a non-cancerous proliferative condition, disorder, or disease. 
     
     
         5 . The process of  claim 1 ,
 wherein the biological sample is obtained from a subject who has an inflammatory and/or degenerative condition, disorder or disease, or who is suspected of having or at risk of having an inflammatory and/or degenerative condition, disorder or disease;   wherein the biological sample is obtained from a subject who has a cardiovascular condition, disorder or disease, or who is suspected of having or at risk of having a cardiovascular condition, disorder or disease.   wherein the biological sample is obtained from a subject who has an infectious condition, disorder or disease, or who is suspected of having or at risk of having an infectious condition, disorder or disease.   
     
     
         6 . The process of  claim 1   comprising (a) isolating or extracting rare cells by passing a biological sample through a polycarbonate filter and recovering the isolated or extracted rare cells on the polycarbonate filter; or.   comprising (a) isolating or extracting rare cells by passing a biological sample through a PET (polyethylene terephthalate) filter and recovering the extracted or isolated rare cells on the PET (polyethylene terephthalate) filter.   
     
     
         7 . A process of  claim 1 , wherein the rare cells isolated or extracted by filtration are transferred to a support before further analyses as in (b) or for culture. 
     
     
         8 . The process of  claim 1 , wherein the isolated or extracted rare cells are cultured prior to (b). 
     
     
         9 . The process of  claim 1 ,
 wherein the isolated or extracted rare cells are fixed prior to (b); or   wherein isolated or extracted rare cells are stained or immunostained on the filter before (b).   
     
     
         10 . The process of  claim 1 , comprising (b) determining the cytomorphology of the isolated or extracted rare cells. 
     
     
         11 . The process of  claim 1 , comprising (b) immunolabeling the isolated rare cells. 
     
     
         12 . The process of  claim 1 , comprising (b) performing molecular analysis on the isolated rare cells. 
     
     
         13 . The process of  claim 1  comprising (b) molecular analysis of the DNA, RNA, or microRNA of the isolated or extracted rare cells. 
     
     
         14 . The process of  claim 1  comprising (b) molecular analysis of the proteins, peptides or polypeptides of the isolated or extracted rare cells. 
     
     
         15 . A process of detection of the presence or absence of rare cells, comprising:
 (a) isolating or extracting rare cells by passing a biological sample through a filter and recovering the isolated rare cells on the filter; wherein the filter has a pore size, pore density or other physical characteristics that retain rare cells but which permit passage of other kinds of cells;   (b) optionally, culturing the isolated or extracted rare cells;   (c) optionally, fixing or staining of the isolated or extracted rare cells or optionally cultured rare cells;   (d) analyzing the isolated or extracted rare cells from (a), (b) or (c) by immunolabeling, and/or in situ molecular analysis, and/or molecular analysis of rare cells DNA, RNA, and/or microRNA, and/or molecular analysis of rare cells protein molecules.   
     
     
         16 . The method of  claim 15 ,
 wherein the isolated or extracted rare cells are lysed and (d) comprises detecting the presence or absence of ALK mutations in the lysed rare cells, wherein said process further comprises selecting a treatment for a subject, following the efficacy of a treatment, or detecting resistance to treatment based on the presence or absence of the ALK mutation;   wherein the isolated or extracted rare cells are lysed and (d) comprises detecting the presence or absence of a K-RAS and/or EGFR mutation in the lysed rare cells, wherein said process further comprises selecting a treatment for a subject, following the efficacy of a treatment, or detecting resistance to treatment based on the presence or absence of the K-RAS and/or EGFR mutation;   wherein the isolated or extracted rare cells are lysed and (d) comprises detecting the presence or absence of a B-RAF and/or HER2 mutation in the lysed rare cells, wherein said process further comprises selecting a treatment for a subject, following the efficacy of a treatment, or detecting resistance to treatment based on the presence or absence of the B-RAF and/or HER2 mutations; or   wherein (d) comprises analyzing the isolated or extracted rare cells by determining the status of epithelial to mesenchymal transition of the rare cells; determining whether the rare cells have a gene-expression signature associated with metastatic or invasive cells; or determining whether the rare cells express determinants associated with metastasis or invasion.   
     
     
         17 . A personalized medicine treatment comprising repeating the process of  claim 15  using biological samples obtained from the same subject at different times;
 wherein the biological samples are obtained from the same patient before and after treatment, at different points during treatment for a condition, or during different treatment regimens for a condition, disorder or disease associated with the rare cells; 
 wherein said personalized treatment may further comprise (e) comparing the number of rare cells between samples obtained a different times to determine efficacy of a treatment regimen or to detect resistance to a treatment regimen, wherein a decrease in the relative number of rare cells detected indicates relative efficacy of a treatment regimen, and wherein an increase in the relative number of rare cells detected indicates resistance to or inefficacy of the treatment regimen; and optionally, 
 (f) selecting an effective personalized targeted treatment for the subject based on (e). 
 
     
     
         18 . The process of  claim 15 , further comprising evaluating an effect of a candidate drug or candidate treatment on molecular characteristics of rare cells, and selecting a drug or treatment that reduces the number of rare cells in a subject compared to a control not given the drug or treatment, and selecting a drug or treatment that reduces the relative number of rare cells or modifies the molecular or immunological characteristics of the rare cells compared to the control. 
     
     
         19 . A composition comprising one or more rare cells isolated or extracted by passing a biological sample through a filter and recovering the isolated rare cells on the filter; wherein the filter has a pore size, pore density or other physical characteristics that retain rare cells but which permit passage of other kinds of cells. 
     
     
         20 . A kit comprising at least one of:
 one or more filters for extracting or isolating rare cells from a biological fluid,   one or more buffers, diluents, or other agents for treating the biological fluid before filtration,   one or more buffers for suspending, washing or otherwise treating rare cells after they are extracted or isolated from a biological fluid,   one or more transfer buffers for transferring the isolated or extracted rare cells from a filter to a different support,   one or more cytomorphological and/or cytochemical staining reagents or other cellular stains, or buffers therefore,   one or more antibodies or other reagents for immunolabeling rare cells or buffers therefore,   one or more reagents for in situ analysis of rare cells on a filter or other support,   one or more lytic agents or lysis buffers for lysing rare cells,   one or more antibodies or other reagents for molecular analysis of rare cell proteins, or buffers therefore,   one or more probes, primers, nucleotides, enzymes or other reagents for molecular analysis of rare cell nucleic acids including PCR.