Single cell pathology analysis of tumour samples

1 . A method of treating a cancer in a patient comprising:

a. acquiring data recorded from a labelled cancer tissue sample with local resolution of a single cell via imaging mass cytometry at a subcellular resolution of ≤5 μm, wherein

the labelled cancer tissue sample is labelled with a plurality of molecular probes, each probe being specific for a biomolecule,

each of said molecular probes is characterized by a detectable marker,

a signal of the detectable marker correlates with an amount of expression of the biomolecule, and

the biomolecules are selected from a list comprising:

i. epithelial cadherin (E-cadherin),

ii. cytokeratin (CK) 18 and/or 19,

iii. CK7,

iv. oestrogen receptor (ER) and/or progesterone receptor (PR),

v. a marker of cellular proliferation,

vi. CK5 and/or p63 and/or CK14,

vii. p53,

viii. a hormone receptor (HR),

ix. a marker of apoptosis,

x. epidermal growth factor receptor (EGFR),

xi. a marker of hypoxia, and

xii. a marker of DNA content,

b. evaluating and processing the data such as to assign each detectable marker to an expression group selected from a high (hi), a positive (+), a low ( lo ), or a negative (−) expression level of the detectable marker in each cell comprised in the labelled cancer tissue sample;

c. assigning each cell a cellular identity (CI), based on the assigned expression group, wherein the CI for each cell is selected from:

CI1: CIAX hi , EGFR−, (ER and/or PR)+, (CK5 and/or p63 and/or CK14)+;

CI2, p53 hi , (cC3 and/or cPARP)+, (ER and/or PR)−, (CK18 and/or CK19)−, (CK5 and/or p63 and/or CK14)−;

CI3, (Ki-67 and/or PCNA)+, CK7−, (CK18 and/or CK19)−, (ER and/or PR)−, (CK5 and/or p63 and/or CK14)−;

CI4, p53 hi , EGFR+, CIAX hi , (ER and/or PR)−, (CK5 and/or p63 and/or CK14)−;

CI5, (CK5 and/or p63 and/or CK14)+, CK7−, (CK18 and/or CK19)−, (ER and/or PR)−;

CI6, E-cadherin hi , (CK18 and/or CK19) hi , CK7+, (ER and/or PR)+, (CK5 and/or p63 and/or CK14)−;

CI7, CK7+, (CK18 and/or CK19)+, (ER and/or PR)−, (CK5 and/or p63 and/or CK14)−;

CI8, E-cadherin-, CK7−, (CK18 and/or CK19)−, (CK5 and/or p63 and/or CK14)−, (ER and/or PR)−;

CI9, (E-cadherin lo or E-cadherin-), ((CK18 and/or CK19)− or (CK18 and/or CK19) lo ), (ER and/or PR) 10 , (CK5 and/or p63 and/or CK14)−;

CI10, ((CK18 and/or CK19) hi or (CK18 and/or CK19)+), E-cadherin+, (ER and/or PR) hi , CK5 and/or p63 and/or CK14)−;

CI11, ((CK18 and/or CK19) hi or (CK18 and/or CK19)+), E-cadherin+, (ER and/or PR)+, CK5 and/or p63 and/or CK14)−;

CI12, (E-cadherin lo or E-cadherin+), ((ER and/or PR) 1° or (ER and/or PR)−), (CK5 and/or p63 and/or CK14)−;

CI13, p53 hi , EGFR+, (ER and/or PR) hi , ((CK5 and/or p63 and/or CK14) lo and/or (CK5 and/or p63 and/or CK14)−);

CI14, CK7+, (CK18 and/or CK19)+, (CK5 and/or p63 and/or CK14)+, (ER and/or PR)−;

d. assigning said cancer tissue sample to a single cell pathology (SCP) patient group according to the proportion of each cellular identity assigned in the cell assignment step the sample contains, wherein the list of SCP patient groups comprises or consists of:

SCP1. >70% of single cells are CI10;

SCP2. >70% of single cells are CI11;

SCP3. ≤70% of single cells are CI10;

SCP4. >70% of single cells to CI12;

SCP5. ≤70% of single cells are CI12;

SCP6. >80% of single cells are CI9;

SCP7. >80% of single cells are CI8;

SCP8. ≤70% of single cells are CI9; or CI10, or CI12;

SCP9. >60% of single cells are CI9;

SCP10. >70% of single cells are CI9; CI10, or CI12;

SCP11. >60% of single cells are CI7;

SCP12. >70% of single cells are CI6;

SCP13. >50% of single cells are CI5;

SCP14. >60% of single cells are CI3;

SCP15. >70% of single cells are CI4;

SCP16. >50% of single cells are CI2;

SCP17. >50% of single cells are CI1;

SCP18. >90% of single cells are CI14;

wherein the patient is assigned to a probable outcome group according to the sample's SCP classification:

SCP1, likely good outcome, and likely sensitive to

a selective oestrogen receptor modulator (SERM) antineoplastic drug;

a selective estrogen receptor degraders (SERD) antineoplastic drug;

an aromatase inhibitor antineoplastic drug; and/or

a PI3K pathway inhibitor drug;

SCP2, likely sensitive to

an antiangiogenic antineoplastic drug; and/or

a HER2 targeting antineoplastic drug;

SCP3, likely poor outcome, likely sensitive to

an anthracycline-type antineoplastic drug;

a mitotic inhibitor-type antineoplastic drug;

an antineoplastic platinum complex;

an alkylating antineoplastic drug;

an antimetabolite-type antineoplastic drug;

a selective SERM antineoplastic drug;

a SERD antineoplastic drug;

an aromatase inhibitor drug; and/or

a PI3K pathway inhibitor drug;

SCP4, likely lack of sensitivity to antineoplastic drugs which target ER

SCP5, likely sensitive to

an EZH2 methyltransferase inhibitor antineoplastic drug;

SCP6, likely poor outcome, likely sensitive

a HER2 targeting antineoplastic drug;

SCP7, likely lack of sensitivity to

an antiangiogenic antineoplastic drug,

and likely sensitive to

a HER2 targeting antineoplastic drug;

SCP8, likely bad outcome;

SCP9, likely sensitive to

a HER2 targeting antineoplastic drug;

SCP10, likely sensitive to

a HER2 targeting antineoplastic drug;

SCP11: likely good outcome, and likely sensitive to

a HER2 targeting antineoplastic drug, and/or

a PI3K pathway inhibitor drug a PI3K pathway inhibitor drug;

SCP12: likely poor outcome, likely sensitive to

an EZH2 methyltransferase inhibitor antineoplastic drug;

SCP13: likely sensitive to

a HER2 targeting antineoplastic drug;

SCP14: likely bad outcome, and likely sensitive to

an anthracycline-type antineoplastic drug;

a mitotic inhibitor-type antineoplastic drug;

an antineoplastic platinum complex;

an alkylating antineoplastic drug; and/or

an antimetabolite-type antineoplastic drug;

SCP15: likely sensitive to

an anthracycline-type antineoplastic drug;

a mitotic inhibitor-type antineoplastic drug;

an antineoplastic platinum complex;

an alkylating antineoplastic drug;

an antimetabolite-type antineoplastic drug; and/or

an inhibitor of EGFR bioactivity antineoplastic drug;

SCP16: likely good outcome, likely sensitive to

an anthracycline-type antineoplastic drug;

a mitotic inhibitor-type antineoplastic drug;

an antineoplastic platinum complex;

an alkylating antineoplastic drug; or

an antimetabolite-type antineoplastic drug;

SCP17: likely bad outcome, and likely sensitive to

a quinone-alkylating antineoplastic drug;

SCP18: likely lack of sensitivity to antineoplastic drugs which target ER, and likely sensitive to a

a PI3K pathway inhibitor drug;

e. administering to the patient an effective amount of a pharmaceutical composition comprising a drug or complex selected from the drugs and complexes to which the SCP is sensitive.

2 . The method of claim 1 , wherein

the SCP is selected from SCP3, SCP14, SCP15, and SCP16; and the drug or complex is selected from

an anthracycline-type antineoplastic drug; or

a mitotic inhibitor-type antineoplastic drug; or

an antineoplastic platinum complex; or

an alkylating antineoplastic drug; or

an antimetabolite-type antineoplastic drug.

3 . The method of claim 1 , wherein

the SCP is SCP1 or SCP3, and the drug or complex is selected from

a selective estrogen receptor modulator (SERM) antineoplastic drug;

a selective estrogen receptor degraders (SERD) antineoplastic drug; and/or

an aromatase inhibitor antineoplastic drug.

4 . The method of claim 1 , wherein the SCP is SCP2, and the drug or complex is an antiangiogenic antineoplastic drug.

5 . The method of claim 1 , wherein the SCP is SCP5 or SCP12, and the drug or complex is an EZH2 methyltransferase inhibitor antineoplastic drug.

6 . The method of claim 1 , wherein the SCP is SCP15, and the drug or complex is an inhibitor of EGFR bioactivity antineoplastic drug.

7 . The method of claim 1 , wherein the SCP is SCP17, and the drug or complex is a quinone-alkylating antineoplastic drug.

8 . The method of claim 1 , wherein the SCP is selected from SCP2, SCP6, SCP 7, SCP 9, SCP 10, SCP11, and SCP13, and the drug or complex is selected from a HER2 targeting antineoplastic drug.

9 . The method of claim 1 , wherein the SCP is selected from SCP1, SCP3, SCP11, and SCP18, and the drug or complex is selected from PI3K pathway inhibitors.

10 . The method according to claim 1 , wherein the method of obtaining information about the average expression of said plurality of biomolecules comprises constructing an image of the tissue sample.

11 . The method according to claim 1 , wherein the method of obtaining information about the average expression of said plurality of biomolecules is imaging mass cytometry at a subcellular resolution of ≤5 μm.

12 . The method according to claim 1 , wherein said method further comprises the steps of:

in step a., including additional markers selected from:

xiii. CD3 or CD90;

xiv. CD20 or CD19;

xiv. CD68;

XV. CD44 and/or CD45;

xvi. Fibronectin;

xvii. vimentin, and

xviii. CD31 and/or von Willibrand factor (vWF) and/or CD34; and

in step c., including additional cellular identities selected from:

CI15. CD44+, CD45+, (CD3 or CD90)+, fibronectin-, E-cadherin-, ((CK5 and/or p63 and/or CK14) 1° or (CK5 and/or p63 and/or CK14)−);

CI16. CD20+, (fibronectin lo or fibronectin-), ((E-cadherin lo or E-cadherin-), ((CK5 and/or p63 and/or CK14) 1° or (CK5 and/or p63 and/or CK14)−);

CI17. (CD3 or CD90)+, (CD20 or CD19)+;

CI18. CD68+;

CI19. vimentin+, (CD34 and/or VWF and/or CD31)+;

CI20. vimentin-, (fibronectin+ or fibronectin hi ), (CD3 or CD90)−, (CD20 or CD19)−, CD45−, CD44−.

13 . The method according to claim 11 , wherein the single cell is a fragment of the image of the tissue sample.

14 . The method according to claim 12 wherein said method further comprises the steps of:

in a cellular community detection step, partitioning the image of the tissue sample into multicellular regions, wherein each single cell inside the multicellular region is highly interconnected to neighbouring cells to provide a cellular community;

in a cellular community assignment step, assigning a cellular community identity (CCI) to each cellular community according to the number of cells in the cellular community, and the proportion of each CI it contains, wherein the list of CCI comprises or consists of:

CCI1. Among cells with identities CI1-CI14, >10% of single cells are CI6, and the average size of cellular communities is >25 cells; or

CCI2. Among cells with identities CI1-CI14, >10% of single cells are CI6, and the average size of cellular communities is ≤25 cells; or

CCI3. Among cells with identities CI1-CI14, >10% of single cells are CI2, and the average size of cellular communities is >25 cells, or

CCI4. Among cells with identities CI1-CI14, >10% of single cells are CI2, and the average size of cellular communities is ≤25 cells, or

CCI5. Among cells with identities CI1-CI14, >10% of cells are CI3, and the average size of cellular communities is ≤25 cells, or

CCI6. >5% of all cells are CI19, and >10% of cells are CI20, and >3% cells are CI18, and the average size of cellular communities is <50 cells, or

CCI7. >80% of all cells are any of the identities CI1 to CI15, and <10% are CI20, and the average size of the cellular communities is <75 cells, or

CCI8. >20% of all cells are CI15, and/or 16 and/or CI17, and <40% cells are any of the identities CI1 to CI14, and the average size of the cellular communities is more than 75 cells, or

CCI9. >5% of all cells are CI18, and the average size of the cellular communities is >25%, or

CCI10 >80% of all cells are any of the identities CI1 to CI14, and <2% cells are CI20, and the average size of the cellular communities is >115 cells and <125 cells,

wherein the patient is assigned to a probable outcome group according to the CCI classification:

CCI1, CCI3, CCI5, CCI8, CCI9, or CCI10: likely good outcome, and

CCI2, CCI4, CCI6, or CCI7: likely bad outcome.