In Vitro Method for the Prognosis of Progression of a Cancer and of the Outcome in a Patient and Means for Performing Said Method

Abstract

The present invention relates to the prognosis of the outcome of a cancer in a patient, which prognosis is based on the quantification of one or several biological markers that are indicative of the presence of, or alternatively the level of, the adaptive immune response of said patient against said cancer.

1 . An in vitro method for the prognosis of survival time of a patient suffering from a cancer, comprising the steps of:
 a) quantifying, in at least one tumor tissue sample from said patient, one or more biological markers indicative of the status of the adaptative immune response of said patient against cancer, said one or more biological markers comprising LAG3;   b) providing predetermined cut-off reference values for LAG3 in tumor tissue samples, said predetermined reference values being correlated with a specific prognosis of progression of survival time;   c) comparing the levels of said one or more biological markers quantified in step a) with the corresponding predetermined reference value provided in step b);   d) determining a prognosis for said patient, wherein:
 a favorable prognosis of survival time is provided for said patient when the quantification values of each of said at least one or more biological markers measured in step a) are, higher than said corresponding predetermined cut-off reference values; and 
 a poor prognosis of survival time is provided for said patient when the quantification values of each of said one or more biological markers are, in both said CT tumor tissue and in said IM tumor tissue, lower than said corresponding predetermined cut-off reference values. 
   
     
     
         2 . The in vitro method of  claim 1 , wherein said one or more biological markers further comprise CD8A. 
     
     
         3 . The in vitro method of  claim 1 , wherein said one or more biological markers further comprise PDCD1 LG1. 
     
     
         4 . The in vitro method of  claim 1 , wherein said one or more biological markers further comprise CD8A and PDCD1 LG1. 
     
     
         5 . The in vitro method of  claim 1 , wherein said step a) of quantifying is performed by quantifying cells of the sample expressing the biological markers. 
     
     
         6 . The in vitro method of  claim 1 , wherein said step a) of quantifying is performed by quantifying the density of cells of the sample expressing the biological markers. 
     
     
         7 . The in vitro method of  claim 1 , said step a) of quantifying includes the step of binding antibodies specifically to the biological markers expressed by cells of the sample. 
     
     
         8 . The in vitro method of  claim 1 , wherein step a) is performed by quantifying at least said one or more biological markers, separately both in the center of the tumor (CT) and in the invasive margin (IM). 
     
     
         9 . The in vitro method of  claim 1 , wherein said step b) of providing cut-off reference values is performed by:
 i) selecting a biological marker for which a reference value is to be determined;   ii) providing a collection of tumor tissue samples from cancer patients by removing one or more pieces or slices of tissue from the tumor, wherein said one or more pieces or slices of tissue include tumor center (CT) tissue from the center of the tumor and invasive margin (IM) tissue from the invasive margin of the tumor;   iii) providing, for each tumor sample provided at step ii), information relating to the actual clinical outcome for the corresponding cancer patient;   iv) providing a series of arbitrary quantification values for the biological marker selected at step i);   v) quantifying the biological marker in each tumor tissue sample contained in the collection provided at step ii);   vi) classifying the tumor samples in two groups for one specific arbitrary quantification value provided at step iii), wherein: (1) a first group comprising tumor samples that exhibit a quantification value for the biological marker that is lower than the arbitrary quantification value contained in the serial of quantification values; and (2) a second group comprising tumor samples that exhibit a quantification value for the biological marker that is higher than the arbitrary quantification value contained in the serial of quantification values; whereby two groups of tumor samples are obtained for the specific quantification value, and wherein the tumors samples of each group are separately enumerated;   vii) calculating the statistical significance between
 the quantification value for the biological marker obtained at step v) and 
 the actual clinical outcome of the patients from which tumor samples contained in the first and second groups defined at step vi) derive; 
   viii) reiterating steps vi) and vii) until every arbitrary quantification value provided, at step iv) is tested;   ix) setting said cut-off reference value as consisting of the arbitrary quantification value for which the highest statistical significance has been calculated at step vii).   
     
     
         10 . The in vitro method according to  claim 1 , wherein the cancer is a solid cancer. 
     
     
         11 . The in vitro method according to  claim 1 , wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, rectum cancer, pancreatic cancer, gastrointestinal carcinoid tumor, stomach cancer, skin cancer, melanoma, lung cancer, bladder cancer, breast cancer, bile duct cancer, laryngeal cancer, hypolaryngeal cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, oral cavity cancer, oropharyngeal cancer, and salivary gland cancer. 
     
     
         12 . The in vitro method according to  claim 1 , wherein the cancer is colorectal cancer. 
     
     
         13 . The in vitro method according to  claim 1 , wherein the survival time is disease-free survival (DFS). 
     
     
         14 . The in vitro method according to  claim 1 , wherein the survival time is overall survival (OS). 
     
     
         15 . The in vitro method according to  claim 1 , further comprising the step of carrying out Tumor-Node-Metastasis (TNM) classification for said patient and providing said TMN classification with said prognosis. 
     
     
         16 . The in vitro method of  claim 1 , further comprising the step of identifying said patient as being at high risk for recurrence of said cancer and likely to benefit from adjuvant therapy when said step of comparing results in provision of a poor prognosis for said patient. 
     
     
         17 . The in vitro method of  claim 1 , wherein
 said method is carried out prior to administering an anticancer agent in order to provide a pre-administration prognosis of survival time; and   said method is carried out after administering said anticancer agent in order to provide a post-administration prognosis of survival time;   and wherein said method further comprises a step of   determining the effectiveness of said anticancer agent by comparing said pre-administration prognosis of survival time with said post-administration prognosis of survival time,   wherein   if said post-administration prognosis of survival time is improved compared to said pre-administration prognosis of survival time, then said anticancer agent is determined to be effective,   and if said post-administration prognosis of survival time is not improved compared to said pre-administration prognosis of survival time,   then said anticancer agent is determined to be ineffective.   
     
     
         18 . A method for treating a patient suffering from a cancer, comprising the steps of:
 a) evaluating the intra-tumor adaptative immune status of said patient before administration of an anti-cancer agent by quantifying, in a pre-administration tumor sample, one or more biological markers indicative of the status of the adaptative immune of said patient against cancer, said one or more biological markers comprising LAG3;   b) administering an anti-cancer agent to the patient;   c) evaluating the intra-tumor adaptative immune status of said patient after administration of said anti-cancer agent by quantifying in a post-administration tumor sample said one or more biological markers indicative of the status of the adaptative immune of said patient against cancer;   d) comparing the levels of said one or more biological markers quantified in step a) with the levels of said at one or more biological markers quantified in step c);   e) administering to the patient:
 an immunotherapeutic agent when the level of LAGS has not increased between step a) and step c). 
   
     
     
         19 . The method of  claim 18 , wherein said one or more biological markers further comprise CD8A. 
     
     
         20 . The method of  claim 18 , wherein said one or more biological markers further comprise PDCD1LG1. 
     
     
         21 . The method of  claim 18 , wherein said one or more biological markers further comprise CD8A and PDCD1LG1. 
     
     
         22 . The method according to  claim 18 , wherein step e) further comprises administering to said patient an adjuvant therapy. 
     
     
         23 . The method according to  claim 18 , wherein said immunotherapeutic agent is an immuno-stimulating cytokine or chemokine. 
     
     
         24 . The method according to  claim 23 , wherein said cytokine or chemokine is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL 7, G-CSF, IL-15, GM-CSF, IFN-γ, CXCL9, CXCL10, Fractalkine, MIG, IFNα, IL-18, IL-12, IL-23 and IL-31. 
     
     
         25 . The method of  claim 24 , wherein said cytokine or chemokine is selected from the group consisting of IL-2, IFNα, IL-7, IL-15, and GM-CSF. 
     
     
         26 . The method of  claim 18 , wherein said pre-administration and post-administration tumor samples include either or both tumor center (CT) tissue from the center of the tumor and invasive margin (IM) tissue of the invasive margin of the tumor. 
     
     
         27 . The method of  claim 26 , wherein said pre-administration and post-administration tumor samples are biopsies. 
     
     
         28 . The method of  claim 18 , wherein the cancer is a solid cancer. 
     
     
         29 . The method of  claim 18 , wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, rectum cancer, pancreatic cancer, gastrointestinal carcinoid tumor, stomach cancer, skin cancer, melanoma, lung cancer, bladder cancer, breast cancer, bile duct cancer, laryngeal cancer, hypolaryngeal cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, oral cavity cancer, oropharyngeal cancer, and salivary gland cancer. 
     
     
         30 . The method of  claim 18 , wherein the cancer is colorectal cancer. 
     
     
         31 . The method according to  claim 18 , wherein said anti-cancer agent is a chemotherapeutic agent.