Method for quantifying immune cells in tumoral tissues and its applications

1. A method for assessment of a number or density of immune cells in tumoral tissues comprising:

a. providing one or more immunostained slides of tissue section obtained by an automated slide-staining system, wherein each slide of tissue section is immunostained by using antibodies binding specifically to markers expressed by immune cells,

b. proceeding to digitalisation of the slides of step a. by high resolution scan capture, whereby a high definition digital picture of the slide to be analysed is obtained,

c. detecting the tissue section on the digital picture,

d. analyzing the slide of tissue section for defining (i) a tumour (CT) and (ii) an invasive margin of the tumour (IM),

e. providing a size reference grid with uniformly distributed units having a same surface, said grid being adapted to the size of the tumour to be analyzed,

f. detecting and quantifying stained cells of the slide whereby the number or the density of immune cells stained of the slide is assessed, then

g. checking the quality of immunostaining by comparison with a reference value, wherein the step g. of checking the quality of immunostaining comprises, for a given marker:

measuring a distribution value representative of a distribution of staining intensities of stained cells in the slide, and

comparing the distribution value with the reference value, the reference value being representative of a staining intensity of stained cells of a reference tissue section having a known distribution of the given marker, the slide being correctly stained if the distribution value is within a range around the reference value and the slide being not correctly stained if the distribution value is not within the range around the reference value, and

h. validating the number or density of stained immune cells of the slide assessed during step f. if the tissue section is correctly stained as checked at step g., or

not validating the number or density of stained immune cells of the slide assessed during step f. if the tissue section is not correctly stained as checked at step g.

2. The method according to claim 1 , wherein detecting and quantifying stained cells of the slide is implemented separately in the tumour (CT) and the invasive margin of the tumour (IM).

3. The method according to claim 1 , wherein the antibodies are selected from the group consisting of antibodies specific for CD3, CD4, CD8, CD20, CD45RO, and GZMB markers.

4. The method according to claim 1 , wherein two or three antibodies selected from the group consisting of the following combinations: CD3+CD8, CD3+CD45RO, CD3+CD20, CD3+GZMB, CD8+CD20, CD8+GZMB, CD8+CD45RO, CD20+GZMB, CD20+CD45RO, GZMB+CD45RO, CD4+CD8, CD4+CD45RO, CD4+GZMB, CD4+CD20, and all the combinations of 3 markers among the CD3, CD8, CD20, CD45RO, CD4 and GZMB markers, are used.

5. The method according to claim 1 , wherein an immunohistochemistry (IHC) automate is used for implementing step a).

6. The method according to claim 1 , wherein step d) comprises analysing the slide of tissue section for defining the boundaries of (i) the tumour (CT) and (ii) the invasive margin of the tumour (IM) by software.

7. The method according to claim 1 , wherein hexagonal, square or rectangular units having a surface of 10×10 −9 m 2 to 1000×10 −9 m 2 are used.

8. The method according to claim 1 , wherein the assessed number or density of immune cells stained of the slide is selected from values of group A:

maximum number of stained cells

maximum density of stained cells

sum of the number of stained cells

sum of the surface of stained cells

sum of the densities of stained cells

mean number of stained cells

mean density of stained cells

maximum number of stained cells and

median of the number of stained cells.

9. The method according to claim 1 , further comprising detecting cells or units according to a criterion selected from Group B:

single stained cells

stained cells in clusters containing at least 3 stained cells

single stained cells or clusters containing at least 3 stained cells

all stained cells

most stained units, wherein said most stained units are a number of units having a highest number of stained cells with respect to other units and

random units.

10. The method according to claim 1 , wherein the reference value is at least one of a mean value, a median value, a minimum value and a maximum value of staining intensity of stained cells of a reference tissue section having a known distribution of the given marker.

11. The method according to claim 1 , wherein the range around the reference value is from 20% below the reference value to 20% above the reference value.

12. The method according to claim 1 , further comprising

provision of an immune score of the tissue section, the provision of an immune score of the tissue section comprising the steps of:

for at least one tumour regions chosen between the tumour (CT) and the invasive margin (IM) of the tumour, comparing a value of the number or the density of immune cells for a given marker with a predetermined threshold value for the given marker

allotting a positive quotation to the tumour region if the value of the number or the density of immune cells is above the predetermined threshold value,

allotting a negative quotation to the tumour region if the value of the number or the density of immune cells is below the predetermined threshold value,

determining the immune score of the tissue section based on a number of positive quotations and a number of negative quotations,

wherein the immune score is a numerical value equal to the number of positive quotations allotted.

13. The method according to claim 12 , wherein the steps of the provision of an immune score are performed for each of the tumour regions.

14. The method according to claim 12 , wherein the steps of the provision of an immune score are performed for a plurality of markers.

15. The method according to claim 12 , further comprising a step of segregating patients into groups according to their immune score, each patient being assigned to one group of patients having the same immune score.

16. The method according to claim 12 , wherein the predetermined threshold value is related to a specific prognosis of progression of said cancer and/or survival of the patient and/or response to a cancer treatment.