Proliferation markers in clinical practice and their use for breast cancer prognosis or diagnosis

Abstract

A method for assessing the proliferative state of cells in a human or non-human biological sample uses Chromatin Assembly Factor-1 (CAF-1) subunits as proliferation markers. The method can be used for breast cancer prognosis or diagnosis, and monitoring tumor response in therapy.

1 . A method for assessing the tumoral state of breast cells in a human or non human biological sample comprising breast cells, wherein said biological sample is suspected of comprising breast tumoral cells, said method comprising the steps of:
 a. detecting and evaluating the expression level of Chromatin Assembly Factor-1 (CAF-1) p60, p150, or p60 and p150 subunits in said biological sample and in a control sample, wherein said control sample (i) comprises healthy breast cells from the same tissue type as said biological sample; (ii) comprises breast tumoral cells from the same tissue type as said biological sample and is characterized by histological grade, or (iii) comprises breast tumoral cells taken prior to treatment from the same tumor and same patient as said biological sample;   b. comparing the CAF-1 subunit expression level in said biological sample with that of said control sample; and   c. correlating (i) a higher CAF-1 subunit expression level in said biological sample as compared to that of said control sample with a higher proliferative state of the breast cells in said biological sample as compared to that of the breast cells in said control sample, or (ii) a lower CAF-1 subunit expression level in said biological sample as compared to that of said control sample with a lower proliferative state of the breast cells in said biological sample as compared to that of the breast cells in said control sample.   
     
     
         2 . The method of  claim 1 , wherein the detection is carried out at the protein level. 
     
     
         3 . The method of  claim 2 , wherein the detection is carried out on phosphorylated CAF-1 p60 subunit. 
     
     
         4 . The method of  claim 2 , wherein the detection is carried out on the total cellular fraction or the chromatin-bound fraction of said CAF-1 subunits, or on the phosphorylated CAF-1 p60 subunit in the cell nucleus. 
     
     
         5 . The method of  claim 2 , wherein the detection is performed by immunofluorescence, Western blot, protein chips, immunocytochemistry or immunohistochemistry. 
     
     
         6 . The method of  claim 5 , wherein said detection involves the use of anti-CAF-1 antibodies, or antibodies targeted against said individual CAF-1 subunits or against fragments thereof, said antibodies being polyclonal or monoclonal antibodies. 
     
     
         7 . The method of  claim 1 , wherein said control sample comprises healthy breast cells from the same tissue type of tissue as said biological sample, and wherein a higher CAF-1 subunit expression level in said biological sample as compared to that of said control sample is correlated with the presence of tumor cells in said biological sample. 
     
     
         8 . The method of  claim 1 , wherein said control sample comprises breast tumoral cells from the same tissue type as said biological sample and is characterized by histological grade, and wherein a higher CAF-1 subunit expression level in said biological sample as compared to that of said control sample is correlated with a higher histological grade in said biological sample as compared to that of said control sample. 
     
     
         9 . The method of  claim 1 , wherein said biological sample comprises breast tumoral cells taken from the breast tumor of a patient after treatment, wherein said control sample comprises breast tumoral cells taken prior to treatment from the same breast tumor and same patient as said biological sample, and wherein a lower CAF-1 subunit expression level in said biological sample as compared to that of said control sample is correlated with an acceptable efficiency of treatment for the patient. 
     
     
         10 . The method of  claim 1 , further comprising detection of Ki-67 or PCNA or MCM or combinations thereof. 
     
     
         11 . The method of  claim 1 , wherein assessing cellular proliferation based on CAF-1 p60, p150, or p60 and p150 subunit expression level is performed by standard Western blot, immunocytochemical and/or immunohistochemical procedures using CAF-1 specific antibodies on cell extracts, fixed cells or tissue sections from a solid breast tumor. 
     
     
         12 . The method of  claim 1 , comprising using a kit for assessing the tumoral state of the breast cells in the human or non human biological sample suspected of comprising breast tumoral cells, said kit comprising a fixative solution for the cell sample to be tested and anti-CAF-1 p60 and/or CAF-1 p150 antibody. 
     
     
         13 . The method of  claim 12 , wherein the kit further comprises a buffer, a blocking agent, a secondary antibody, reagents to prepare an avidin-biotin complex and/or a counter-staining solution. 
     
     
         14 . The method of  claim 12 , wherein the kit further comprises coverslips or slides. 
     
     
         15 . A method for assessing the survival prognostic of a patient with breast cancer comprising the steps of:
 a. detecting and evaluating the expression level of Chromatin Assembly Factor-1 (CAF-1) p60, p150, or p60 and p150 subunits in a human or non human biological sample suspected of comprising breast tumoral cells;   b. comparing the CAF-1 subunit expression level in said biological sample with that of a cut off value; and   c. correlating a CAF-1 subunit expression level in said biological sample below said cut off value with a higher patient survival prognostic.