Method of scoring a sample comprising tumor tissue

文档序号：1859865 发布日期：2021-11-19 浏览：6次中文

阅读说明：本技术 对包含肿瘤组织的样品评分的方法 (Method of scoring a sample comprising tumor tissue ) 是由詹尼弗·波尔德奥克斯纳温·达卡帕加里泰·特兰布莱恩·利特尔金周荣于 2016-10-21 设计创作，主要内容包括：本发明部分涉及对包含取自癌症患者的肿瘤组织的样品进行评分的方法。该分值表示至少一对细胞之间的空间接近度,所述至少一对细胞的第一成员表达第一生物标志物,并且所述至少一对细胞的第二成员表达不同于第一生物标志物的第二生物标志物。从这些方法获得的分值可以指示患者可能会对免疫治疗积极应答的似然度。(The present invention relates in part to methods of scoring a sample comprising tumor tissue taken from a cancer patient. The score represents a spatial proximity between at least one pair of cells, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker. The scores obtained from these methods may indicate the likelihood that the patient is likely to respond positively to the immunotherapy.)

1. A method of scoring a sample comprising tumor tissue taken from a cancer patient, comprising:

(i) determining a score representing spatial proximity between at least one pair of cells using the sample comprising tumor tissue taken from the cancer patient, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker; and

(ii) recording the score.

2. The method of claim 1, wherein the first member of the at least one pair of cells comprises a tumor cell and the second member of the at least one pair of cells comprises a non-tumor cell.

3. The method of claim 2, wherein the non-tumor cell comprises an immune cell.

4. The method of claim 1, wherein the first member and the second member of the at least one pair of cells comprise immune cells.

5. The method of claim 1, wherein the first member of the at least one pair of cells comprises a tumor cell, a bone marrow cell, or a stromal cell, and the second member of the at least one pair of cells comprises an immune cell.

6. The method of claim 5, wherein the tumor cell, the bone marrow cell, or the stromal cell expresses PD-L1, and the immune cell expresses PD-1.

7. The method of claim 1, wherein the spatial proximity is evaluated on a pixel scale.

8. The method of claim 1, wherein the spatial proximity between the at least one pair of cells is in a range of 1 pixel to 100 pixels.

9. The method of claim 1, wherein the spatial proximity between the at least one pair of cells is in the range of 0.5 μ ι η to 50 μ ι η.

10. The method of claim 1, wherein the determining step comprises:

(i) selecting a predetermined number of fields of view obtainable from the sample comprising tumor tissue stained with a plurality of fluorescent markers taken from the cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing the first biomarker than other fields of view;

(ii) (ii) for each selected field of view, enlarging the fluorescence signal attributable to the first biomarker by a margin sufficient to encompass proximally located cells expressing the second biomarker; and

(iii) dividing a first total area of all cells in each selected field of view expressing the second biomarker and encompassed within the expanded fluorescence signal attributable to the cell expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score.

11. The method of claim 10, wherein each of the fluorescent labels is specific for a particular biomarker.

12. The method of claim 10, wherein the plurality of fluorescent labels comprises a first fluorescent label for the first biomarker and a second fluorescent label for the second biomarker.

13. The method of claim 10, wherein the margin is in a range of 1 pixel to 100 pixels.

14. The method of claim 10, wherein the proximally located cells expressing the second biomarker are within 0.5 to 50 μ ι η of a plasma membrane of the cells expressing the first biomarker.

15. The method of claim 10, wherein the first total area is measured in pixels.

16. The method of claim 10, wherein the normalization factor is a second total area of all non-tumor cells in each selected field of view.

17. The method of claim 10, wherein the normalization factor is a second total area of all cells in each selected field of view that have the ability to express the second biomarker.

18. The method of claim 16, wherein the second total area is measured in pixels.

19. The method of claim 10, wherein the predetermined factor is 10⁴。

20. The method of claim 1, wherein the first member of the at least one pair of cells expresses a first biomarker selected from the group consisting of PD-L1, PD-L2, B7-H3, B7-H4, HLA-DR, galectin 9, CD80, CD86, 4.1BBL, ICOSL, CD40, OX40L, IDO-1, GITRL, and combinations thereof, and the second member of the at least one pair of cells expresses a second biomarker selected from the group consisting of PD-1, TIM3, LAG3, 41BB, OX40, CTLA-4, CD40L, CD28, GITR, ICOS, CD28, and combinations thereof.

21. The method of claim 20, wherein the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses PD-1.

22. The method of claim 20, wherein the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses CD 80.

23. The method of claim 20, wherein the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 80.

24. The method of claim 20, wherein the first member of the at least one pair of cells expresses PD-L2 and the second member of the at least one pair of cells expresses PD-1.

25. The method of claim 20, wherein the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 86.

26. The method of claim 20, wherein the first member of the at least one pair of cells expresses LAG-3 and the second member of the at least one pair of cells expresses HLA-DR.

27. The method according to claim 20, wherein said first member of said at least one pair of cells expresses TIM-3 and said second member of said at least one pair of cells expresses galectin 9.

28. The method of claim 20, wherein the first member of the at least one pair of cells expresses 41BB and the second member of the at least one pair of cells expresses 4.1 BBL.

29. The method of claim 20, wherein the first member of the at least one pair of cells expresses OX40 and the second member of the at least one pair of cells expresses OX 40L.

30. The method of claim 20, wherein the first member of the at least one pair of cells expresses CD40 and the second member of the at least one pair of cells expresses CD 40L.

31. The method of claim 20, wherein the first member of the at least one pair of cells expresses ICOS and the second member of the at least one pair of cells expresses ICOSL.

32. The method of claim 20, wherein the first member of the at least one pair of cells expresses GITR and the second member of the at least one pair of cells expresses GITRL.

33. The method of claim 20, wherein the first member of the at least one pair of cells expresses HLA-DR and the second member of the at least one pair of cells expresses TCR.

34. The method of claim 1, further comprising comparing the score to a threshold.

35. The method of claim 34, wherein the threshold range is 500 to 5000.

36. The method of claim 35, wherein the threshold is 900 plus or minus 100.

37. The method of claim 1, wherein the immunotherapy comprises immune checkpoint therapy.

38. A method of determining a score representing a spatial proximity between at least one pair of cells selected from a plurality of cells present in a predetermined number of fields of view obtainable from a sample comprising tumor tissue taken from a cancer patient, the method comprising:

(i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased towards selecting fields of view comprising a greater number of cells expressing a first specific biomarker than other fields of view;

(ii) (ii) for each selected field of view, enlarging the fluorescence signal attributable to the first specific biomarker by a margin sufficient to encompass proximally located cells expressing a second specific biomarker; and

(iii) dividing a first total area of all cells in each selected field of view expressing the second specific biomarker and covered within the expanded fluorescence signal attributed to the cell expressing the first specific biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score.

39. The method of claim 38, wherein each of the fluorescent labels is specific for a particular biomarker.

40. The method of claim 38, wherein the plurality of fluorescent labels comprises a first fluorescent label for the first specific biomarker and a second fluorescent label for the second specific biomarker.

41. The method of claim 38, wherein the one or more fluorescent labels comprise a fluorophore conjugated to an antibody having binding affinity for a particular biomarker or another antibody.

42. The method of claim 38, wherein each fluorescent marker comprises a fluorophore selected from one or more of DAPI, Cy2, Cy3, 5, Cy5, FITC, TRITC, 488 dye, 555 dye, 594 dye, and Texas Red.

43. The method of claim 38, wherein the margin is in a range of 1 pixel to 100 pixels.

44. The method of claim 38, wherein the proximally located cells expressing the second specific biomarker are within 0.5 to 50 μ ι η of the plasma membrane of the cells expressing the first specific biomarker.

45. The method of claim 38, wherein the first total area is measured in pixels.

46. The method of claim 38, wherein the normalization factor is a second total area of all non-tumor cells in each selected field of view.

47. The method of claim 38, wherein the normalization factor is a second total area of all cells in each selected field of view that have the ability to express the second specific biomarker.

48. The method of claim 46, wherein the second total area is measured in pixels.

49. The method of claim 38, wherein the predetermined factor is 10⁴。

50. The method of claim 38, wherein the spatial proximity score, SPS, is determined by the equation:

wherein A is_IIs the total interaction area which isThe total area of cells expressing the second specific biomarker and covered by an expanded fluorescence signal attributed to cells expressing the first specific biomarker, Ac is the total area of cells having the ability to express the second specific biomarker.

51. The method of claim 38, wherein the first specific biomarker comprises tumor and non-tumor markers and the second specific biomarker comprises non-tumor markers.

Background

The present invention relates generally to the field of cancer therapy.

Disclosure of Invention

Disclosed herein in one aspect is a method of scoring a sample comprising tumor tissue taken from a cancer patient, comprising: (i) determining a score representing spatial proximity between at least one pair of cells using a sample comprising tumor tissue taken from a cancer patient, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker; and (ii) recording a score that, when compared to a threshold value, indicates the likelihood that the cancer patient will respond positively to the immunotherapy. In some embodiments, the first member of the at least one pair of cells comprises a tumor cell and the second member of the at least one pair of cells comprises a non-tumor cell. In some embodiments, the non-tumor cells comprise immune cells. In some embodiments, the first member and the second member of the at least one pair of cells comprise immune cells. In some embodiments, the first member of the at least one pair of cells comprises a tumor cell, a bone marrow cell, or a stromal cell, and the second member of the at least one pair of cells comprises an immune cell. In some embodiments, the tumor cells, bone marrow cells, or stromal cells express PD-L1, and the immune cellsExpressing PD-1. In some embodiments, spatial proximity is evaluated on a pixel scale. In some embodiments, the spatial proximity between at least one pair of cells is in the range of about 1 pixel to about 100 pixels. In some embodiments, the spatial proximity between at least one pair of cells is in the range of about 0.5 μm to about 50 μm. In some embodiments, the determining step comprises: (i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing a first biomarker than other fields of view; (ii) for each selected field of view, enlarging the fluorescence signal attributable to the first biomarker by a margin sufficient to encompass proximally located cells expressing the second biomarker; and (iii) dividing the first total area of all cells in each selected field of view that express the second biomarker and are encompassed within the expanded fluorescence signal attributable to the cells expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a (arrive at) spatial proximity score. In some embodiments, each of the fluorescent labels is specific for a particular biomarker. In some embodiments, the plurality of fluorescent labels includes a first fluorescent label for the first biomarker and a second fluorescent label for the second biomarker. In some embodiments, the margin is in a range of about 1 pixel to about 100 pixels. In some embodiments, the proximally located cells expressing the second biomarker are within about 0.5 to about 50 μm of the plasma membrane of the cells expressing the first biomarker. In some embodiments, the first total area is measured in units of pixels. In some embodiments, the normalization factor is a second total area of all non-tumor cells in each selected field of view. In some embodiments, the normalization factor is a second total area of all cells in each selected field of view that have the ability to express the second biomarker. In some embodiments, the normalization factor is a second total area of all cells in each selected field of view. In some embodiments, the second total area is measured in units of pixels. In some embodiments, the reservation isFactor is 10⁴. In some embodiments, the first member of the at least one pair of cells expresses a first biomarker selected from the group consisting of PD-L1, PD-L2, B7-H3, B7-H4, HLA-DR, galectin 9, CD80, CD86, 4.1BBL, ICOSL, CD40, OX40L, IDO-1, GITRL, and combinations thereof, and the second member of the at least one pair of cells expresses a second biomarker selected from the group consisting of PD-1, TIM3, LAG3, 41BB, OX40, CTLA-4, CD40L, CD28, GITR, ICOS, CD28, and combinations thereof. In some embodiments, the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses PD-1. In some embodiments, the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses CD 80. In some embodiments, the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 80. In some embodiments, the first member of the at least one pair of cells expresses PD-L2 and the second member of the at least one pair of cells expresses PD-1. In some embodiments, the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 86. In some embodiments, a first member of the at least one pair of cells expresses LAG-3 and a second member of the at least one pair of cells expresses HLA-DR. In some embodiments, the first member of the at least one pair of cells expresses TIM-3 and the second member of the at least one pair of cells expresses galectin 9. In some embodiments, the first member of the at least one pair of cells expresses 41BB and the second member of the at least one pair of cells expresses 4.1 BBL. In some embodiments, the first member of the at least one pair of cells expresses OX40 and the second member of the at least one pair of cells expresses OX 40L. In some embodiments, the first member of the at least one pair of cells expresses CD40 and the second member of the at least one pair of cells expresses CD 40L. In some embodiments, the first member of the at least one pair of cells expresses ICOS and the second member of the at least one pair of cells expresses ICOSL. In some embodiments, the first member of the at least one pair of cells expresses GITR and the second member of the at least one pair of cellsThe member expresses GITRL. In some embodiments, a first member of the at least one pair of cells expresses HLA-DR and a second member of the at least one pair of cells expresses TCR. In some embodiments, the threshold range is about 500 to about 5000. In some embodiments, the threshold is about 900 +/-100. In some embodiments, the immunotherapy comprises an immune checkpoint therapy. In some embodiments, the method provides superior predictive power compared to quantitatively estimating the expression of the first biomarker or quantitatively estimating the expression of the second biomarker. In some embodiments, predictive power is quantified as a positive predictive value, a negative predictive value, or a combination thereof. In some embodiments, the positive predictive value is 65% or greater. In some embodiments, the positive predictive value is 70% or greater. In some embodiments, the positive predictive value is 75% or greater. In some embodiments, the negative predictive value is 65% or greater. In some embodiments, the negative predictive value is 80% or greater.

Disclosed herein in another aspect is a method of determining a score representing the spatial proximity between at least one pair of cells selected from a plurality of cells present in a predetermined number of fields of view obtainable from a sample comprising tumor tissue taken from a cancer patient, the method comprising: (i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased towards selecting fields of view comprising a greater number of cells expressing a first specific biomarker than other fields of view; (ii) (ii) for each selected field of view, enlarging the fluorescence signal attributable to the first specific biomarker by a margin sufficient to encompass proximally located cells expressing the second specific biomarker; and (iii) dividing the first total area of all cells in each selected field of view that express the second specific biomarker and are encompassed within the expanded fluorescent signal attributable to the cells expressing the first specific biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score. In some embodiments, each of the fluorescent labels is specific for a particular biomarker. In some embodiments, a plurality of fluorescent marker packetsComprising a first fluorescent label for a first biomarker and a second fluorescent label for a second biomarker. In some embodiments, the one or more fluorescent labels comprise a fluorophore conjugated to an antibody or another antibody having binding affinity for a particular biomarker. In some embodiments, each fluorescent marker comprises a fluorophore independently selected from one or more of the group consisting of DAPI, Cy2, Cy3, 5, Cy5, FITC, TRITC, 488 dye, 555 dye, 594 dye, and Texas Red (Texas Red). In some embodiments, the margin is in a range of about 1 pixel to about 100 pixels. In some embodiments, the proximally located cells expressing the second specific biomarker are within about 0.5 to about 50 μm of the plasma membrane of the cells expressing the first specific biomarker. In some embodiments, the first total area is measured in units of pixels. In some embodiments, the normalization factor is a second total area of all non-tumor cells in each selected field of view. In some embodiments, the normalization factor is a second total area of all cells in each selected field of view that have the ability to express a second particular biomarker. In some embodiments, the normalization factor is a second total area of all cells in each selected field of view. In some embodiments, the second total area is measured in units of pixels. In some embodiments, the predetermined factor is 10⁴. In some embodiments, the Spatial Proximity Score (SPS) is determined by the following equation:

wherein A is_IIs the total interaction area (total area of cells expressing the second specific biomarker and covered by an expanded fluorescence signal attributable to the cells expressing the first specific biomarker), A_cIs the total area of cells having the ability to express the second specific biomarker. In some embodiments, the method provides superior predictive power compared to quantitatively estimating expression of the first biomarker or quantitatively estimating expression of the second biomarker. In some embodiments, predictive power is quantified as a positive predictive value, a negative predictive value, or a combination thereof. In some embodiments, the positive predictive value is 65% or greater. In some embodiments, the positive predictive value is 70% or greater. In some embodiments, the positive predictive value is 75% or greater. In some embodiments, the negative predictive value is 65% or greater. In some embodiments, the negative predictive value is 80% or greater.

In some embodiments, the first specific biomarker comprises tumor and non-tumor markers and the second specific biomarker comprises a non-tumor marker.

Drawings

FIG. 1 shows a non-limiting example of a profile of antibodies and detection reagents used in preparing tissue samples for imaging and analysis.

FIG. 2a shows a non-limiting example of all nuclei detected with DAPI within an image.

FIG. 2b shows a non-limiting example of an expanded binary mask of all cells within the image of FIG. 2 a.

Fig. 3a shows a non-limiting example of an image of S100 detected with 488 dye.

FIG. 3b shows a non-limiting example of a binary mask of all tumor regions within FIG. 3 a.

FIG. 3c shows a non-limiting example of a mask for all tumor cells within FIG. 3 a.

FIG. 3d shows a non-limiting example of a mask for all non-tumor cells within FIG. 3 a.

FIG. 4a shows a non-limiting example of an image of PD-L1 detected with Cy5.

FIG. 4b shows a non-limiting example of a binary mask for all PD-L1 positive cells within FIG. 4 a.

FIG. 5a shows a non-limiting example of an image of PD-1 detected with Cy ® 3.5.

FIG. 5b shows a non-limiting example of a binary mask of all PD-1 positive non-tumor cells within FIG. 5 a.

FIG. 6a shows a non-limiting example of an interaction mask for all PD-L1-positive cells and the nearest neighbor cells.

FIG. 6b shows a non-limiting example of the interaction compartment (interaction component) of PD-1 positive cells in close proximity to PD-L1 positive cells.

Figure 7a shows a non-limiting example of the interaction scores for 26 melanoma patients.

FIG. 7b shows a non-limiting example of the maximum interaction score for the 26 patients of FIG. 7 a.

Fig. 8 shows the results of an analysis based on whole-slice imaging instead of the enrichment algorithm.

Fig. 9 shows the interaction score for 26 patients compared to progression free survival (survival). Note that: indicates uncorrected log-rank test.

Figure 10 shows a comparison of PD-L1 expression with progression free survival of patients.

FIG. 11 shows a non-limiting example of a mask of fluorescence signals corresponding to PD-L1 positive cells (red), PD-1 positive cells (yellow), all tumor cells (green), and all cells that are positive responders to immunotherapy (blue).

FIG. 12 shows a non-limiting example of a mask of fluorescence signals corresponding to PD-L1 positive cells (red), PD-1 positive cells (yellow), all tumor cells (green), and all cells that are negative responders to immunotherapy (blue).

FIG. 13 shows representative PD-1/PD-L1 interaction scores for 38 non-small cell lung cancer patients.

Fig. 14 is a flowchart of a process for scoring a sample including tumor tissue, according to an exemplary embodiment.

Fig. 15 is a flowchart of a process for scoring a sample containing tumor tissue according to the second exemplary embodiment.

Fig. 16 is a block diagram of a controller configured to score a sample containing tumor tissue taken from a cancer patient, according to an exemplary embodiment.

Fig. 17 is a flowchart of image processing steps for scoring a sample containing tumor tissue, according to an exemplary embodiment.

Figure 18 shows a comparison of PD-L1 expression determined using the 22C3 FDA-approved IHC assay with progression-free survival of patients. Note: indicated p values were determined using an uncorrected log rank test.

Figure 19a shows a non-limiting example of interaction scores from an additional 34 melanoma patients.

Figure 19b shows a comparison of the interaction score of the patient of figure 19a with progression-free survival.

Figure 19c shows the interaction scores for the patient from figure 7 and the patient of figure 19 a.

Figure 19d shows a comparison of the interaction score of the patient of figure 19c with progression-free survival. Note: indicated p values were determined by using an uncorrected log rank test.

Figure 19e shows a comparison of the interaction score with Overall Survival (OS) for the patient of figure 19 c. Note: indicated p values were determined by using an uncorrected log rank test.

Figure 20 shows a non-limiting example of CTLA-4/CD80 interaction scores from 29 metastatic melanoma patients.

FIG. 21 shows a non-limiting example of PD-1/PD-L1 interaction scores from 29 patients with testicular cancer.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Various embodiments are described below. It should be noted that these specific embodiments are not intended as an exhaustive description or as a limitation on the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and may be practiced with any other embodiment(s).

As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which the term is used. If there is a use of a term that is not clear to one of ordinary skill in the art, "about" will mean up to plus or minus 10% of the particular term, given the context in which the term is used.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments and does not pose a limitation on the scope of the claims unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The terms "treatment" or "treating" refer to administering a treatment in an amount, manner, or mode that is statistically significant or detectable to a person of skill in the art to effectively ameliorate a condition, symptom, or parameter associated with a disease or to prevent worsening of a disease. The effective amount, mode or pattern may vary from patient to patient and may be tailored to the patient.

Disclosed herein in one aspect is a method of scoring a sample comprising tumor tissue taken from a cancer patient.

In some embodiments, the sample may be stained with a plurality of fluorescent labels having affinity for a particular biomarker. A digital image of the stained sample may be acquired and the image may be further analyzed based on the location of the fluorescent marker. Rather than performing a full image analysis, the field of view may be prioritized based on the number of cells expressing the first biomarker of interest. A predetermined number of fields of view may then be further analyzed for fluorescence signals. In some embodiments, four different types of fluorescent labels are used to generate an image of the fluorescent signal corresponding to a first biomarker of interest and an image of the fluorescent signal corresponding to a second biomarker of interest, as well as an image of the fluorescent signals corresponding to biomarkers expressed by all cells and an image of the fluorescent signal corresponding to biomarkers expressed by tumor cells. In further embodiments, the image of the fluorescence signals is manipulated to generate one or more masks of fluorescence signals corresponding to cells within the image. In some embodiments, the one or more masks of the fluorescence signal comprise one or more selected from the group consisting of: a mask of all cells within the image, a mask of all tumor cells within the image, a mask of all non-tumor cells within the image, a mask of all cells within the image that express the first biomarker of interest, a mask of all cells within the image that express the second biomarker of interest, and an interaction mask that represents all cells within the image that express the first biomarker of interest and cells located nearby that express the second biomarker of interest. In yet another embodiment, the interaction mask is used to generate interaction compartments of cells expressing the second biomarker of interest in all selected fields of view that are in proximity to cells expressing the first biomarker of interest. The total area of the interaction compartments can be used to generate a score representing the spatial proximity between at least one pair of cells, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker. In some embodiments, the score is indicative of a score of the likelihood that the cancer patient will respond positively to the immunotherapy. In some embodiments, the method provides superior predictive power compared to quantitatively estimating the expression of a first biomarker of interest or quantitatively estimating the expression of a second biomarker of interest.

Thus, in some embodiments, provided herein are methods of scoring a sample comprising tumor tissue taken from a cancer patient, comprising: (i) determining a score representing spatial proximity between at least one pair of cells using a sample comprising tumor tissue taken from a cancer patient, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker; and (ii) recording a score that, when compared to a threshold value, indicates the likelihood that the cancer patient will respond positively to the immunotherapy. In some embodiments, the method provides superior predictive power compared to quantitatively estimating the expression of the first biomarker or quantitatively estimating the expression of the second biomarker.

In some embodiments, the first member of the at least one pair of cells comprises a tumor cell and the second member of the at least one pair of cells comprises a non-tumor cell. In some embodiments, the non-tumor cell is an immune cell. In some embodiments, the non-tumor cell is a stromal cell.

In some embodiments, the first member and the second member of the at least one pair of cells comprise immune cells.

In some embodiments, the first member of the at least one pair of cells comprises a tumor cell, a bone marrow cell, or a stromal cell, and the second member of the at least one pair of cells comprises an immune cell. In some embodiments, the tumor cells, bone marrow cells, or stromal cells express PD-L1 and the immune cells express PD-1.

In some embodiments, the first member of the at least one pair of cells comprises a tumor cell and the second member of the at least one pair of cells comprises an immune cell. In some embodiments, the first member of the at least one pair of cells comprises bone marrow cells and the second member of the at least one pair of cells comprises immune cells. In some embodiments, the first member of the at least one pair of cells comprises stromal cells and the second member of the at least one pair of cells comprises immune cells. In some embodiments, the first member of the at least one pair of cells expresses PD-L1 and the immune cell expresses PD-1.

In some embodiments, the first member of the at least one pair of cells expresses a first biomarker selected from the group consisting of PD-L1, PD-L2, B7-H3, B7-H4, HLA-DR, galectin 9, CD80, CD86, 4.1BBL, ICOSL, CD40, OX40L, IDO-1, GITRL, and combinations thereof. In some embodiments, the second member of the at least one pair of cells expresses a second biomarker selected from the group consisting of PD-1, TIM3, LAG3, 41BB, OX40, CTLA-4, CD40L, CD28, GITR, ICOS, CD28, and combinations thereof. In some embodiments, the first member of the at least one pair of cells expresses a first biomarker selected from the group consisting of PD-L1, PD-L2, B7-H3, B7-H4, HLA-DR, galectin 9, CD80, CD86, 4.1BBL, ICOSL, CD40, OX40L, IDO-1, GITRL, and combinations thereof, and the second member of the at least one pair of cells expresses a second biomarker selected from the group consisting of PD-1, TIM3, LAG3, 41BB, OX40, CTLA-4, CD40L, CD28, GITR, ICOS, CD28, and combinations thereof.

In some embodiments, the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses PD-1. In some embodiments, the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses CD 80. In some embodiments, the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 80. In some embodiments, the first member of the at least one pair of cells expresses PD-L2 and the second member of the at least one pair of cells expresses PD-1. In some embodiments, the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 86. In some embodiments, a first member of the at least one pair of cells expresses LAG-3 and a second member of the at least one pair of cells expresses HLA-DR. In some embodiments, the first member of the at least one pair of cells expresses TIM-3 and the second member of the at least one pair of cells expresses galectin 9. In some embodiments, the first member of the at least one pair of cells expresses 41BB and the second member of the at least one pair of cells expresses 4.1 BBL. In some embodiments, the first member of the at least one pair of cells expresses OX40 and the second member of the at least one pair of cells expresses OX 40L. In some embodiments, the first member of the at least one pair of cells expresses CD40 and the second member of the at least one pair of cells expresses CD 40L. In some embodiments, the first member of at least one pair of cells expresses ICOS and the second member of the at least one pair of cells expresses ICOSL. In some embodiments, the first member of the at least one pair of cells expresses GITR and the second member of the at least one pair of cells expresses GITRL. In some embodiments, the first member of the at least one pair of cells expresses HLA-DR and the second member of the at least one pair of cells expresses TCR.

In some embodiments, a first biomarker expressed by a first member of the at least one pair of cells and a second biomarker expressed by a second member of the at least one pair of cells interact with each other. In some embodiments, the first biomarker expressed by the first member of the at least one pair of cells and the second biomarker expressed by the second member of the at least one pair of cells do not interact with each other.

In some embodiments, the spatial proximity between at least one pair of cells is in the range of about 0.5 μm to about 50 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 50 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 45 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 40 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 35 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 30 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 25 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 20 μm. In some embodiments, the spatial proximity is in the range of 2.5 μm to about 15 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 50 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 45 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 40 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 35 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 30 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 25 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 20 μm. In some embodiments, the spatial proximity is in the range of 5 μm to about 15 μm. In some embodiments, the spatial proximity is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μm.

In some embodiments, the spatial proximity between at least one pair of cells is in the range of about 1 pixel to about 100 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 100 pixels. In some embodiments, the spatial proximity ranges from about 5 pixels to about 90 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 80 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 70 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 60 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 50 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 40 pixels. In some embodiments, the spatial proximity is in the range of about 5 pixels to about 30 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 100 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 90 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 80 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 70 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 60 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 50 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 40 pixels. In some embodiments, the spatial proximity is in the range of about 10 pixels to about 30 pixels. In some embodiments, the spatial proximity is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pixels. In some embodiments, the pixel width is 0.5 μm.

In some embodiments, the determining step comprises: (i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing a first biomarker than other fields of view; (ii) for each selected field of view, enlarging the fluorescence signal attributable to the first biomarker by a margin sufficient to encompass proximally located cells expressing the second biomarker; and (iii) dividing the first total area of all cells in each selected field of view that express the second biomarker and are encompassed within the expanded fluorescence signal attributable to the cells expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score.

In some embodiments, the determining step comprises: (i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing a first biomarker than other fields of view; (ii) (ii) for each selected field of view, expanding the fluorescence signal attributable to the first biomarker to encompass cells expressing the second biomarker located nearby within about 0.5 μm to about 50 μm of the plasma membrane of the cells expressing the first biomarker; and (iii) dividing the first total area of all cells in each selected field of view that express the second biomarker and are encompassed within the expanded fluorescence signal attributable to the cells expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score.

In some embodiments, the determining step comprises: (i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing a first biomarker than other fields of view; (ii) for each selected field of view, expanding the fluorescence signal attributable to the first biomarker by a margin ranging from about 1 pixel to about 100 pixels to encompass proximally located cells expressing the second biomarker; and (iii) dividing a first total area of all cells in each selected field of view measured in pixels that express the second biomarker and are encompassed within the expanded fluorescence signal attributable to the cells expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score.

In some embodiments, the determining step comprises: (i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing a first biomarker than other fields of view; (ii) expanding the fluorescence signal attributable to the first biomarker by a margin ranging from about 1 pixel to about 100 pixels for each selected field of view to encompass cells expressing the second biomarker within about 0.5 μm to about 50 μm of the plasma membrane of the cells expressing the first biomarker; and (iii) dividing a first total area of all cells in each selected field of view measured in pixels that express the second biomarker and are encompassed within the expanded fluorescence signal attributable to the cells expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor to arrive at a spatial proximity score.

In some embodiments, four fluorescent labels are used in the determining step, wherein each fluorescent label is specific for a different biomarker. In further embodiments, the first fluorescent label is associated with a first biomarker, the second fluorescent label is associated with a second biomarker, the third fluorescent label is associated with a third biomarker, and the fourth fluorescent label is associated with a fourth biomarker. In some embodiments, the first biomarker comprises tumor and non-tumor markers. In some embodiments, the second biomarker comprises a non-tumor marker. In some embodiments, the first biomarker comprises tumor and non-tumor markers, and the second biomarker comprises non-tumor markers. In some embodiments, the third biomarker is expressed by all cells. In some embodiments, the fourth biomarker is expressed only in tumor cells. In some embodiments, the third biomarker is expressed by all cells, and the fourth biomarker is expressed only in tumor cells. In some embodiments, the one or more fluorescent labels comprise a fluorophore conjugated to an antibody or another antibody having binding affinity for a particular biomarker. In some embodiments, the one or more fluorescent labels are fluorophores with affinity for a particular biomarker.

Examples of fluorophores include, but are not limited to, fluorescein (fluorscein), 6-FAM, rhodamine (Rhabdamine), Texas Red (Texas Red), California Red (California Red), iFluor594, tetramethylrhodamine (tetramethylrhodamine), carboxyrhodamine (Carboxyrhodamine) C, carboxyrhodamine 6F (carboxyrhodamine 6F), carboxyrhodamine (carboxyrhodamine) C, carboxyrhodamine 110 (carboxyrhodamine 110), Cascafe Blue, Cascade Yellow, coumarin (coumarin), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy-Chrome, DyLight 350, Light, Dyhrin, Cy 405, Cy 55, Cy 3975, Cy5.5, Cy 55, Cy-5, Cy-David black tea No. 55, Cy-5-C, Cy-Daphb-5, Cy-5-C13, Cy-5-C, Cy-5-C.5, Cy-Daphb.5, Cy-C.5, Cy-C.405, Cy-C.9, Cy-C.5, Cy-C.9, Cy-C.5, Cy-C.9, Cy-C.C.C.9, Cy-C.9, Cy-C.C.C.C.9, Cy II, Cy II, Cy-C.C.9, Cy II, Cy-C.9, Cy-C.9, Cy-C.9, Cy II, Cy 9, Cy II, Cy 9, Cy 9, Cy 9, Cy II, Cy 9, Cy II, Cy 9, Cy, NED, ROX (5- (and-6-) -carboxyl-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alex Fluor 430, Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, 7-amino-4-methylcoumarin TR-3-acetic acid, BODIPY 570, BODIPY 2, BODIPY 2, BODIPY G, BODIPY 630/650, BODIPY 3655, BODIPY 3665, BODIPY 369, OPAL ™ 690 and combinations thereof. In some embodiments, the fluorophore is selected from the group consisting of DAPI, Cy2, Cy3, Cy5, Cy7, FITC, TRITC, 488 dye, 555 dye, 594 dye, Texas Red, and coumarin. Examples of 488 dyes include, but are not limited to, Alexa Fluor488, DyLight 488, and CF 488A. Examples of 555 dyes include, but are not limited to, Alexa Fluor 555. Examples of 594 dyes include, but are not limited to, Alexa Fluor 594.

As used herein, a "field of view" refers to a portion of a digital image of an entire slice of a tissue sample. In some embodiments, the entire slice image has 2-200 predetermined fields of view. In some embodiments, the entire slice image has 10-200 predetermined fields of view. In some embodiments, the entire slice image has 30-200 predetermined fields of view. In some embodiments, the entire slice image has 10-150 predetermined fields of view. In some embodiments, the entire slice image has 10-100 predetermined fields of view. In some embodiments, the entire slice image has 10-50 predetermined fields of view. In some embodiments, the entire slice image has 10-40 predetermined fields of view. In some embodiments, the entire slice image has 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 (including an increased number thereof) predetermined fields of view.

In some embodiments, the fluorescence signal attributable to the first biomarker is amplified by a margin ranging from about 1 pixel to about 100 pixels. In some embodiments, the margin is about 5 pixels to about 100 pixels. In some embodiments, the margin is about 5 pixels to about 90 pixels. In some embodiments, the margin is about 5 pixels to about 80 pixels. In some embodiments, the margin is about 5 pixels to about 70 pixels. In some embodiments, the margin is about 5 pixels to about 60 pixels. In some embodiments, the margin is about 5 pixels to about 50 pixels. In some embodiments, the margin is about 5 pixels to about 40 pixels. In some embodiments, the margin is about 5 pixels to about 30 pixels. In some embodiments, the margin is about 10 pixels to about 100 pixels. In some embodiments, the margin is about 10 pixels to about 90 pixels. In some embodiments, the margin is about 10 pixels to about 80 pixels. In some embodiments, the margin is about 10 pixels to about 70 pixels. In some embodiments, the margin is about 10 pixels to about 60 pixels. In some embodiments, the margin is about 10 pixels to about 50 pixels. In some embodiments, the margin is about 10 pixels to about 40 pixels. In some embodiments, the margin is about 10 pixels to about 30 pixels. In some embodiments, the margin is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 pixels. In some embodiments, the pixel width is 0.5 μm.

In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 0.5 μm to about 50 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 50 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 45 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 40 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 35 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 30 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 25 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 20 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 2.5 μm to about 15 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 50 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 45 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 40 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 35 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 30 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 25 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 20 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located nearby within about 5 μm to about 15 μm of the plasma membrane of the cell expressing the first biomarker. In some embodiments, the fluorescent signal attributable to the first biomarker is expanded to encompass cells expressing the second biomarker located within about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μm of the plasma membrane of cells expressing the first biomarker. In some embodiments, the second biomarker located on a nearby cell is in direct contact with the first biomarker.

In some embodiments, the first total area of all cells expressing the second biomarker in each selected field of view is measured in pixels.

In some embodiments, the normalization factor is a second total area of all non-tumor cells in each selected field of view. In some embodiments, the second total area is measured in pixels. In some embodiments, the first total area and the second total area are both measured in pixels.

In some embodiments, the normalization factor is a second total area of all cells in each selected field of view that have the ability to express the second biomarker. In some embodiments, the second total area is measured in pixels. In some embodiments, the first total area and the second total area are both measured in pixels.

In some embodiments, the normalization factor is a second total area of all cells in each selected field of view. In some embodiments, the second total area is measured in pixels. In some embodiments, the first total area and the second total area are both measured in pixels.

In some embodiments, the threshold score is about 500 to about 5000. In some embodiments, the threshold score is about 500 to about 4500. In some embodiments, the threshold score is about 500 to about 4000. In some embodiments, the threshold score is about 500 to about 3500. In some embodiments, the threshold score is about 500 to about 3000. In some embodiments, the threshold score is about 500 to about 2500. In some embodiments, the threshold score is about 500 to about 2000. In some embodiments, the threshold score is about 500 to about 1500. In some embodiments, the threshold score is about 500 to about 1000. In some embodiments, the threshold score is about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000, including incremental numbers therein. In some embodiments, the threshold score is about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000, including increments of plus or minus 100 therein.

In some embodiments, the predetermined factor is about 10 to about 10⁵. In some embodiments, the predetermined factor is about 10²To about 10⁵. In some embodiments, the predetermined factor is about 10³To about 10⁵. In some embodiments, the predetermined factor is about 10⁴To about 10⁵. In some embodiments, the predetermined factor is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10⁵Including an increased number therein.

In some embodiments, predictive power is quantified as a positive predictive value, a negative predictive value, or a combination thereof. Positive predictive values were calculated by dividing the number of patients responding to treatment with scores above a threshold score by the total number of patients responding to treatment. Negative predictive values were calculated by dividing the number of patients who did not respond to treatment with a score below the threshold score by the total number of patients who did not respond to treatment.

In some embodiments, the positive predictive value is greater than 60%. In some embodiments, the positive predictive value is 65% or greater. In some embodiments, the positive predictive value is 70% or greater. In some embodiments, the positive predictive value is 75% or greater. In some embodiments, the positive predictive value is 80% or greater. In some embodiments, the positive predictive value is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, including increasing numbers therein.

In some embodiments, the negative predictive value is 60% or greater. In some embodiments, the negative predictive value is 65% or greater. In some embodiments, the negative predictive value is 70% or greater. In some embodiments, the negative predictive value is 75% or greater. In some embodiments, the negative predictive value is 80% or greater. In some embodiments, the negative predictive value is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, including increasing numbers therein.

In the methods disclosed herein, the cancer patient is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is not a human. In further embodiments, the mammal is a mouse, rat, guinea pig, dog, cat, or horse.

In the methods disclosed herein, the tumor tissue is taken from a cancer patient. The types of cancer include, but are not limited to: cancers of the circulatory system, for example, cancers of the heart (sarcomas [ angiosarcomas, fibrosarcomas, rhabdomyosarcomas, liposarcomas ], myxomas, rhabdomyomas, fibromas, lipomas, and teratomas), cancers of the septum and pleura, and cancers of other internal thoracic organs, vascular tumors, and tumor-associated vascular tissue; cancers of the respiratory tract (e.g., nasal and middle ear, paranasal sinuses, larynx, trachea, bronchi and lung), such as Small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), bronchial carcinomas (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; cancers of the digestive system, for example, cancers of the esophagus (e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), abdomen (carcinoma, lymphoma, leiomyosarcoma), stomach, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, open bowel peptide tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract cancers, for example, cancers of the kidney (adenocarcinoma, Wilm's tumor [ nephroblastoma ], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); cancers of the liver, e.g., hepatocellular carcinoma (hepatocellular carcinoma), cholangiocellular carcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (e.g., pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor, and glucagonoma); cancers of the bone (e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteochondral exostosis), benign chondroma, chondroblastoma, osteogenic-like osteoma, and giant cell tumor); cancers of the nervous system, e.g., tumors of the Central Nervous System (CNS), primary CNS lymphomas, cancers of the head (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), cancers of the meninges (meningioma, meningosarcoma, brain glioma), brain cancers (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [ pinealoma ], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma; cancers of the reproductive system, for example, cancers of the gynecological aspect, of the uterus (endometrial cancer), of the cervix (cervical cancer, pre-cervical lesions), of the ovary (ovarian cancer [ serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma ], granulocytic tumors, Leydig cell tumors, dysgerminomas, malignant teratomas), of the vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), of the vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma)), of the fallopian tubes (carcinoma), and of other sites associated with female reproductive organs; cancers of the placenta, penis, prostate, testis, and other parts associated with the male reproductive organs; cancers of the hematological system, e.g., cancers of the hematological (myelogenous leukemia [ acute and chronic ], acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), hodgkin's disease, non-hodgkin's lymphoma [ malignant lymphoma ]; oral cancers, such as those of the oral cavity, e.g., the lips, tongue, gums, floor of the mouth, palate, parotid gland and other parts of the salivary glands, tonsils, oropharynx, nasopharynx, pyriform fossae, throat, and other parts of the lips, oral cavity, and pharynx; cancers of the skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, nevus dysplastic nevus, lipoma, hemangioma, cutaneous fibroma, and keloid; cancers of the adrenal gland: neuroblastoma; and cancers of other tissues including connective and soft tissues, retroperitoneal cavity and peritoneum, melanoma and adnexa of the eye, breast, head and/or neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and non-specific malignant lymph node tumors, secondary malignancies of the respiratory and digestive systems and secondary malignancies of other sites, or one or more combinations thereof.

Examples of immunotherapy include, but are not limited to, monoclonal antibodies (e.g., alemtuzumab or trastuzumab), conjugated monoclonal antibodies (e.g., ibritumomab, brentuximab vendottin, or trastuzumab antibody-drug conjugates), bispecific monoclonal antibodies (bornauzumab), immune checkpoint inhibitors (e.g., yiprimumab, pembrobamizumab, nilutamab, tivumab, atezolizumab, or duvacizumab), thalidomide, lenalidomide, pomamadamide, and imiquimod, and combinations thereof. In some embodiments, the immunotherapy comprises an immune checkpoint therapy.

In some embodiments, the Spatial Proximity Score (SPS) is determined by the following equation:

wherein A is_IIs the total interaction area (total area of cells expressing the second specific biomarker and covered by the amplified fluorescent signal attributed to the cell expressing the first specific biomarker), and A_NTIs the total area of non-tumor cells.

In some embodiments, the Spatial Proximity Score (SPS) is determined by the following equation:

wherein A is_IIs the total interaction area (total area of cells expressing the second specific biomarker and covered by the amplified fluorescent signal attributed to the cells expressing the first specific biomarker), A_cIs the total area of cells having the ability to express the second specific biomarker.

In another aspect, a method of scoring a sample comprising tumor tissue from a cancer patient is used in a method of treating cancer in a patient. In some embodiments, the method of scoring a sample comprising tumor tissue from a cancer patient is performed prior to administration of immunotherapy.

In another aspect, disclosed herein is a method of treating cancer in a patient in need thereof, the method comprising: (a) scoring a sample comprising tumor tissue taken from a patient, comprising (i) determining a score representing spatial proximity between at least one pair of cells using a sample comprising tumor tissue taken from a patient, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker; and (ii) recording the score; (b) comparing the score to a threshold; and (c) applying immunotherapy to the patient if the score, when compared to a threshold, indicates a likelihood that the cancer patient will respond positively to the immunotherapy. In some embodiments, the determining step is as described herein.

Figure 14 is a flow chart depicting the steps of one embodiment of a method for scoring a sample comprising tumor tissue taken from a cancer patient. In step 1401, image data is obtained, and in step 1402, the image data is unmixed so that data specific to various types of fluorescence signals are separated into different channels. In step 1403, data from the first channel is used to generate a mask for all cells that are positive for the first biomarker (first biomarker mask). The masks for all cells are then expanded (step 1404) to generate an expanded mask that represents the predetermined proximity in which the interacting cells (positive for the second biomarker) can be found. In some embodiments, the first biomarker mask is expanded by a pixel value between 1 and 100 pixels. In step 1405, data from the second channel is used to generate a mask for all cells that are positive for the second biomarker (second biomarker mask). In step 1406, the first biomarker mask and the second biomarker mask are combined to generate an interaction mask that identifies cells that are positive for the second biomarker within a predetermined proximity of cells that are positive for the first biomarker. In step 1407, a spatial proximity score is calculated based on the area of the interaction mask.

Fig. 15 is a second flow chart depicting the steps of a second embodiment of a method for scoring a sample comprising tumor tissue taken from a cancer patient. In step 1501, image data is acquired and in step 1502, the image data is unmixed such that data specific to various types of fluorescence signals are separated into different channels. In step 1503, data from the first channel is used to generate a mask for all cells in the field of view, and in step 1504, data from the second channel is used to generate a mask for a subset of regions (e.g., tumor regions) in the field of view. In step 1505, the masks for all cells are combined with the subset area mask to generate a mask for the subset cells and a mask for the non-subset cells. In some embodiments, the subset of cells are tumor cells and the non-subset of cells are non-tumor cells. In step 1506, data from the third channel is used to generate a mask for all cells that are positive for the first biomarker (first biomarker mask). The mask of all positive cells is then expanded (step 1507) to generate an expanded mask representing the predetermined proximity in which the interacting cells (positive for the second biomarker) can be found. In some embodiments, the first biomarker mask is expanded by a pixel value between 1 and 100 pixels. In step 1508, the data from the fourth channel is used to generate a mask for all cells that are positive for the second biomarker (second biomarker mask). In step 1509, the dilated mask and the second biomarker mask are combined to generate an interaction mask that identifies cells positive for the second biomarker within a predetermined proximity of cells positive for the first biomarker. In step 1510, a spatial proximity score is calculated by dividing the area of the interaction mask by the area of all cells (subset of cells) that are capable of being positive for the second biomarker or by the area of all cells (as indicated by the use of either input in the dashed line in the flowchart of fig. 15). In some embodiments, the cell that is capable of being positive for the second biomarker is a tumor cell or a non-tumor cell.

In some embodiments, the subset of cells and the non-subset of cells correspond to tumor cells and non-tumor cells, respectively, and vice versa. In some embodiments, the subset of cells and the non-subset of cells correspond to viable cells and non-viable cells, respectively, and vice versa. In some embodiments, the subset of cells is a subset of living cells, and the non-subset of cells consists of living cells that are not comprised in the subset of living cells. In some embodiments, the subset cells and non-subset cells correspond to T cells and non-T cells, respectively, and vice versa. In some embodiments, the subset cells and non-subset cells correspond to bone marrow cells and non-bone marrow cells, respectively, and vice versa.

In some embodiments, the spatial proximity score represents the proximity of a pair of cells. In some embodiments, the closeness of a pair of cells is determined by proximity between their boundaries, proximity between their centroids, using boundary logic based on a perimeter around a selected first cell of the pair, determining an intersection of their boundaries, and/or by determining an overlapping area of the pair of cells.

In some embodiments, the spatial proximity score is associated with metadata associated with an image of the sample, included in the generated report, provided to an operator to determine an immunization instruction policy, recorded in a database, associated with a medical record of the patient, and/or displayed on a display device.

In the methods disclosed herein, according to an exemplary embodiment, the manipulation of the digital image may be performed by a computing system including a controller, such as the controller shown in the block diagram of fig. 16. The controller 200 is shown to include a communication interface 202 and a processing circuit 204. The communication interface 202 may include a wired or wireless interface (e.g., jack, antenna, transmitter, receiver, transceiver, wire connection, etc.) for data communication with various systems, devices, or networks. For example, the communication interface 202 may include an ethernet card and port for transmitting and receiving data via an ethernet-based communication network and/or a WiFi transceiver for communicating via a wireless communication network. Communication interface 202 may be configured to communicate via a local or wide area network (e.g., the internet, a building WAN, etc.) and may use various communication protocols (e.g., BACnet, IP, LON, etc.).

The communication interface 202 may be a network interface configured to facilitate electronic data communication between the controller 200 and various external systems or devices (e.g., the imaging device 102). For example, the controller 200 may receive imaging data for a selected field of view from the imaging device 102 for analysis of the data and calculation of a Spatial Proximity Score (SPS).

Still referring to fig. 16, the processing circuit 204 is shown to include a processor 206 and a memory 208. The processor 206 may be a general or special purpose processor, an Application Specific Integrated Circuit (ASIC), one or more Field Programmable Gate Arrays (FPGAs), a set of processing components, or other suitable processing components. The processor 506 may be configured to execute computer code or instructions stored in the memory 508 or received from other computer-readable media (e.g., CDROM, network storage, remote server, etc.).

The memory 208 may include one or more devices (e.g., memory units, memory devices, storage devices, etc.) for storing data and/or computer code for accomplishing and/or facilitating the various processes described in this disclosure. Memory 208 may include Random Access Memory (RAM), Read Only Memory (ROM), hard drive storage, temporary storage, non-volatile memory, flash memory, optical storage, or any other suitable memory for storing software objects and/or computer instructions. The memory 208 may include database components, object code components, script components, or any other type of information structure for supporting the various activities and information structures described in this disclosure. Memory 508 may be communicatively connected to processor 206 via processing circuitry 204 and may include computer code for executing (e.g., by processor 206) one or more processes described herein.

Still referring to fig. 16, the controller 200 is shown receiving input from the imaging device 102. The imaging device acquires all imaging data and will record it and all metadata describing it. The imaging device then serializes the data into a stream that can be read by the controller 200. The data stream may accommodate any binary data stream type, e.g., file system, RDBM, or direct TCP/IP communication. For use of the data stream, the controller 200 is shown to include a spectral unmixer 210. The spectral unmixer 210 may receive image data from the imaging device 102, perform spectral unmixing on the image data to unmix images exhibiting various wavelengths to separate discrete channels for each band. For example, the image data may be "unmixed" into separate channels for each of the various fluorophores used to identify cells or proteins of interest in the tissue sample. By way of example only, the fluorophore may be one or more of the group consisting of DAPI, Cy2, Cy3, 5, Cy5, FITC, TRITC, 488 dye, 555 dye, 594 dye, and Texas red. In one example, one of the channels may include image data falling within a predetermined band of wavelengths around 461nm (the maximum emission wavelength for DAPI) to identify nuclei in the image. Other channels may include different wavelengths of image data to identify different portions of the tissue sample using different fluorophores.

Controller 200 is also shown to include various masks, such as cell mask 212, subgroup area mask 216, first biomarker mask 222, and second biomarker mask 224. In other embodiments, these or other maskers, which may be included in the controller 200, are used to receive the unmixed signals from the spectral unmixer 210 and create masks for particular cells or regions of interest from fluorophores used to identify certain features of interest in the tissue sample. To create the mask, the masks (e.g., cell mask 212, subgroup area mask 216, first biomarker mask 222, and second biomarker mask 224) receive image data relating to the intensity of each pixel in the field of view. The pixel intensity is proportional to the amount of fluorescence emitted by the sample, which in turn is proportional to the amount of protein biomarker in the sample (when a fluorophore is used to identify a particular biomarker). The absolute threshold may be set based on the values present in the image pixels. All pixels greater than or equal to the threshold will be mapped to 1.0 or "on" and all other pixels will be mapped to 0.0 or "off". In this manner, a binary mask is created to identify the cell or tissue portion of interest in the field of view. In other embodiments, a lower limit is used to create a mask, where all pixels with intensities at or above the lower limit are accepted and used as pixel values for the mask. If the intensity is below the lower limit, the pixel value is set to 0.0 or "none".

In the example flow chart for masking shown in fig. 17, it is shown that the DAPI and 488 dye channels (used to identify nuclei and tumor regions, respectively) provide the masking output using a lower limit scheme (steps 1710, 1712, 1720, step 1722), while the Cy5 and Cy3.5 channels (used to identify biomarkers) use a threshold scheme (steps 1730, 1740). Associated with the lower bound approach is the further need for a histogram step to determine the lower bound. In particular, it is possible to use, for example,histogram threshold(step 1712, step 1722) a threshold value for the input image is generated and a floating scale (sliding scale) is used to determine the point at which the threshold value occurs. The input being current image and user definedPercentage threshold. The latter is used to determine on which percentage of the total intensity the threshold level should be set. First, the intensity of each pixel is added to the total intensity.Percentage thresholdMultiplying by the total intensity to obtainThe cut-off sum (cut-off sum) is obtained. Finally, all pixels are grouped by intensity (histogram) and their intensities are added from lowest to highest (column-by-column) until a truncated sum is reached. The last highest pixel intensity visited in the process is the threshold for the current image. All pixels with intensities greater than this value have their intensities set to maximum and all other pixels are set to minimum.

The steps identified in FIG. 17 as step 1714, step 1716, step 1724, step 1726, step 1728, step 1732, step 1734, step 1736, step 1742, step 1744 represent intermediate steps that occur in the initial maskers (e.g., cell masker 212, subgroup area masker 216, first biomarker masker 222, and second biomarker masker 224). These steps are defined as follows:

enlargementThe area of the brightest region in the image is increased. To is directed atEnlargementTwo inputs are required. The first being an implicit current picture and the second being to be enlargedIterationThe number of (2). Assume that only binary images are used for the first input. If the program operates on successive images, the output will not be validEnlargement。EnlargementThe process first finds the maximum pixel intensity in the image. Subsequently, each pixel in the image is examined once. If the intensity of the pixel under examination is equal to the maximum intensity, the pixel will be rendered in the output image as havingIterationA circle of radius and centered on the original pixel. The intensity of all pixels in the circle is equal to the maximum intensity. All other pixels will be copied directly into the output image without modification.

Hole filling processThe "blank" areas of the image may be filled with the pixels of maximum intensity. These empty regions are those with the least intensity and pixel area (Size and breadth) Is an area designated by the user. Current image sumSize and breadthTwo inputs are required. LikeEnlargementThe process should be applied only to binary images.

Etching ofTo and withEnlargementThe image is processed in the same manner. In a first step, in the imageOnly the pixel matching the lowest intensity is modified and the circle used to enlarge the found pixel of the lowest intensity is filled with the lowest intensity value, except for the remaining functions andenlargementThe same is true. LikeEnlargementThe process should be applied only to binary images.

Removing objects. Two inputs are expected: current image sumObject size。Removing objectsAndhole filling processThe opposite is true. Fill area containing only maximum intensity is smaller than inputObject sizeAny area of the pixel of (a) will be set to the minimum intensity and thus "removed". This process should be applied only to binary images; application to successive images may yield undesirable results.

The outputs at final step 1718, step 1729, step 1738, and step 1746 are the resulting cell mask, the subgroup area mask (or, in this particular example, the tumor area mask), the biomarker 1 cell mask, and the biomarker 2 cell mask, respectively. FIG. 17 further depicts the combination of these resulting masks to calculate a spatial proximity score. These combinations are described below with reference to the combination masker of controller 200 depicted in fig. 16.

Controller 200 is shown to include combination masks, e.g., subset cell mask 218, non-subset cell mask 220, and interaction mask 230. Subset cell mask implementationAnd（And) The output of cell mask 212 (representing all cells in the image) is operated (as shown in step 1752 in fig. 17) to be combined with the output of subgroup area mask 216. Thus, the subset cell mask generates a mask of all subset cells in the image. In some embodiments, the subset of cells are tumor cells. Using a mask performed by a non-subset cell mask 220Output of（out) The same combination of operations (as shown in step 1754 in fig. 17) generates a mask for all non-subset cells in the sample image. In some embodiments, the non-subset of cells are non-tumor cells.

The first biomarker mask (from the first biomarker mask 222) is dilated by the dilator 226 prior to combining with another mask. The dilation mask represents an area surrounding those cells expressing the first biomarker in order to identify the space of cells expressing the second biomarker that will be within the proper proximity to interact with the cells expressing the first biomarker. This is represented by steps 1756 and 1758 of FIG. 17. The flow chart of fig. 17 shows that the expansion occurs in two steps, 1756 and 1758. When there is a limit to the maximum iteration in each step, it may be necessary to achieve dilation in both steps. For example, a maximum of 10 iterations may be allowed (corresponding to a 10 pixel increase), so when a 20 pixel increase is required, the expansion must be split into two subsequent steps.

Within the second biomarker mask 224, one may useAnd (And)Operation biomarker masks are combined with the non-subset masks described above (as shown in step 1760 of fig. 17) to generate a mask for all non-subset cells that are positive for the first biomarker. The mask is then combined with the expanded mask from the expander 226 at the interaction mask 230 (step 1762) to generate an interaction mask. The interaction mask identifies non-subset cells that are positive for the second biomarker and that are also within the interaction region or overlap with the expanded mask. These identified cells then represent cells that can interact with cells positive for the first biomarker, thereby achieving a stronger therapeutic response.

To calculate the Spatial Proximity Score (SPS), the area of the interaction mask is determined in pixels at the area evaluator 232. In some embodiments, the area of all cells capable of expressing the second biomarker is determined in pixels at the area evaluator 234. The cell capable of expressing the second biomarker may be a tumor cell or a non-tumor cell. In some embodiments, the area of all cells is determined in pixels in area evaluator 234. The interaction or spatial proximity score is determined at interaction calculator 236 by dividing the area from area evaluator 232 by the area from area evaluator 234 multiplied by a predetermined factor. As described above, in one embodiment, the interaction calculator 236 runs the equation:

wherein A is_IIs the total interaction area (total area of cells expressing the second specific biomarker and covered within the expanded fluorescence signal attributable to the cells expressing the first specific biomarker), A_CIs the total area of cells having the ability to express the second specific biomarker or the total area of all cells in the field of view.

In twoAND (AND)After operation pairAndThe process models, but differs in important respects.AndThe current image and the user selected result are accepted. The output is an image created by performing a multiplication between the normalized intensities of the matched pixels from the two input images. In some cases, the image intensity data has been normalized. Therefore, the temperature of the molten metal is controlled,Andthe process is simply a pixel-by-pixel multiplication between the two images.OutThe two inputs required are the current image and the result selected by the user. According to formula A (1-B/B)_max) Removing the second image from the first image, wherein A is the current image, B is the image selected by the user for removal, and B is the image selected by the user for removal_maxIs the maximum intensity of B. Note that B is divided by B_maxB is normalized.

Examples of the invention

Example 1. Sample preparation, imaging, and imaging analysis of melanoma tissue samples from human patients

Sample preparation. Formalin Fixed Paraffin Embedded (FFPE) tissue samples were deparaffinized. The sections were then rehydrated through a series of xylenes before incubation in distilled water and washed with alcohol. Heat-induced antigen retrieval was then performed under conditions of elevated pressure and temperature, followed by cooling and transferring to Tris (Tris-hydroxymethyl-aminomethane) buffered saline. The dyeing is then carried out, in which the following steps are carried out. First, endogenous peroxidase was blocked to reduce prior to incubation with protein blocking agentNon-specific antibody staining. Next, sections were stained with murine anti-PD 1 primary antibody (primary antibody). The sections were then washed prior to incubation with anti-murine HRP secondary antibody (secondary antibody). PD-1 staining was detected using TSA + Cy3.5 (Perkin Elmer) after the sections were washed. Any residual HRP was then quenched by two washes with fresh 100mM benzoyl hydrazine containing 50mM hydrogen peroxide. Sections were washed again before staining with rabbit anti-PD-L1 primary antibody. After sections were washed, incubation was performed with a mixture of anti-rabbit HRP secondary antibody and murine anti-S100 directly labeled with 488 dye and 4', 6-diamidino-2-phenylindole (DAPI). After washing the sections again, PD-L1 staining was detected using TSA-Cy 5 (Perkin Elmer). The sections were washed for the last time, and then coverslips with mounting medium were covered on the sections and dried overnight at room temperature. Figure 1 shows a schematic overview of antibodies and detection reagents. Alternatively, the anti-CD 8 primary antibody was used in place of the anti-PD 1 primary antibody to stain the sections.

Sample imaging and analysis. Fluorescence images were then acquired using a Vectra 2 smart slice analysis system (using Vectra software version 2.0.8 (perkin elmer)). First, the slices were monochromatic imaged at 4x (4 x) magnification using DAPI. An automated algorithm (developed with inForm) is used to identify regions in the slice that contain tissue.

For the channels associated with DAPI (blue), FITC (green), and Cy5 (red), the identified tissue-containing slice regions were imaged at 4x magnification to create RGB images. These 4x magnification images were processed using an automated enrichment algorithm (developed with inForm) in the field of view selector 104 to identify and arrange the possible 20x magnification fields according to the highest Cy @ 5 expression.

The largest 40 fields of view were imaged at 20x magnification of DAPI, FITC, Texas Red, and Cy5 wavelengths. The raw images were examined for acceptability and images that were unfocused, absent any tumor cells, highly necrotic, or contained fluorescent signals that were highly unrelated to the expected antibody localization (i.e., background staining) were excluded prior to analysis. The received images are processed using AQUAduct (perkin elmer), where each fluorophore is spectrally unmixed to a single channel by spectral unmixer 210 and saved as a separate file.

The processed files were further analyzed using AQUAnalysis ™ or by a fully automated process using AQUAserver ™. Details are as follows.

Each DAPI image is processed by the cell mask 212 to identify all nuclei within the image (fig. 2 a), and then the image is enlarged by 3 pixels to represent the approximate size of the entire cell. The resulting mask represents all cells within the image (fig. 2 b).

S100 (tumor cell marker for melanoma) detected with 488 dye was processed by tumor mask 216 (fig. 3 a) to create a binary mask of all tumor regions within the image (fig. 3 b). The overlap between this mask and the mask of all cells creates a new mask for the tumor cells by using the tumor cell mask 218 (fig. 3 c).

Similarly, the absence of tumor cell markers in combination with the mask of all nuclei creates a new mask for all non-tumor cells (fig. 3 d), a process performed by non-tumor cell masker 220.

Each Cy5 image (FIG. 4 a) is processed by the first biomarker mask 222 and overlaps with the mask of all cells to create a binary mask of all cells positive for PD-L1 (FIG. 4 b). Overlaying the biomarker mask with the masks for all cells eliminates noise pixels in the masks that may be falsely identified as biomarker positive cells.

Each Cy3.5 image (FIG. 5 a) is processed through the second biomarker mask 224 to create a binary mask of PD-1 positive cells and overlapped with the masks of all non-tumor cells to create a binary mask of non-tumor cells that are PD-1 positive (FIG. 5 b). Overlaying the biomarker mask with the masks for all non-tumor cells eliminates noise pixels in the masks that may be falsely identified as biomarker positive cells.

The binary mask of all PD-L1 positive cells is expanded using the second expander 226 to create an interaction mask that encompasses the nearest neighbor cells (e.g., cells with PD-1) (fig. 6 a). This interaction mask was combined with a binary mask of all PD-1 positive non-tumor cells by using interaction mask 230 to create an interaction compartment of PD-1 positive cells that was very close to PD-L1 positive cells such that PD-1 could potentially interact with PD-L1 (fig. 6 b).

The total area for all acceptable regions of the interaction compartment (up to 40 fields of view) and the total area of non-tumor cells were calculated in the area assessors 232, 234, respectively. The total area of all acceptable fields of view from the interaction compartment was divided by the total area of non-tumor cells and multiplied by a factor of 10000 by using the interaction calculator 236 to create a complete number representing the interaction score for each sample. The PD-L1 and PD-1 measurements have high reproducibility (R respectively)²=0.98 and 0.97). A broader range of PD-L1 and PD-1 expression and interaction scores were observed in archived clinical samples (n = 53). In a population of 26 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 21), the PD-1/PD-L1 interaction score was found to be able to distinguish responders from non-responders (p = 0.01), but PD-L1 alone (p = 0.07) or CD8 alone (p = 0.23) did not. In addition, patients who showed higher PD-1/PD-L1 interaction scores had higher response rates (82% vs 20%, p = 0.01). Patients with higher PD-1/PD-L1 interaction scores experienced longer progression-free survival (p = 0.059) and lower mortality (22% vs 58%) than patients with lower PD-1/PD-L1 interaction scores. These results indicate that this method of scoring tissue samples to obtain a PD-1/PD-L1 interaction score provides superior predictive power (82% positive predictive value, 80% negative predictive value) compared to PD-L1 expression alone.

Representative scores for 26 patients are shown in fig. 7A. Based on these data, a threshold of 800-.

Optionally, an interaction score is calculated for each individual field of view, and the maximum score for each patient is shown in fig. 7 b. Based on the maximum score, a threshold 1900 is selected to represent the likelihood of response to treatment.

To evaluate the effect of the enrichment algorithm on the interaction score, the above process was performed using whole-slice imaging instead of the enrichment algorithm (see fig. 8). When whole-slice imaging was performed, there was no statistically significant difference between patients who responded to the anti-PD 1 treatment and those who did not respond. Therefore, the threshold cannot be determined by this analysis.

The interaction scores were compared to Progression Free Survival (PFS) of the patients (figure 9). An interaction score of at least 803 exhibits good correlation with survival. Notably, PD-L1 expression was not associated with improved PFS (fig. 10).

Fig. 11 and 12 show representative examples of overlapping masks indicating PD-L1 positive cells (red), PD-L-positive cells (yellow), tumor cells (S100, green), and all cells (DAPI, blue). For positive responders to immunotherapy, the mask in FIG. 11 readily indicated the presence of PD-L1 positive cells (red), PD-1 positive cells (yellow), and all tumor cells (green). In contrast, for negative responders to immunotherapy, the mask in fig. 12 indicated the presence of tumor cells (S100, green) and all cells (DAPI, blue), but hardly PD-L1 positive cells (red) or PD-1 positive cells (yellow). Figure 11 represents an interaction score (complete response to immunotherapy) with a value of 2176. Figure 12 represents an interaction score (no response to immunotherapy) of 8.

Tissue samples were also evaluated using FDA approved methods to detect PD-L1 in non-small cell lung cancer with anti-PD-L1 antibody clone 22C3, but have not been used for melanoma tissue samples so far. PD-L1 expression was compared to patient PFS and is shown in figure 18. This method does not show statistically relevant diagnostic values compared to the method using interaction scores described herein.

A test population of 34 additional metastatic melanoma patients was examined and a PD-1/PD-L1 interaction score was obtained (see figure 19 a). These interaction scores were also compared to the Progression Free Survival (PFS) of the patients (fig. 19 b). Although not statistically significant (p = 0.19), this comparison indicates that patients with higher PD-1/PD-L1 interaction scores have a tendency to have longer PFS. Since the clinical use of these therapies is relatively recent, statistical significance may be limited, thus limiting the follow-up time for these patients.

The PD-1/PD-L1 interaction scores for the combination of the 26 early patient population and the 34 test patient population and the comparison of these scores to the patient PFS or Overall Survival (OS) of the patient are shown in fig. 19 c-19 e. Analysis of the combination clearly indicated that patients with higher PD-1/PD-L1 showed better response to anti-PD-1 treatment.

EXAMPLE 2 sample preparation, imaging, and imaging analysis of non-Small cell Lung cancer tissue samples from human patients

A similar procedure to example 1 was performed, wherein 488-dye-directly labeled murine anti-pan cytokeratin (mouse anti-pan cytokeratin) was used in place of 488-dye-directly labeled murine anti-S100 for epithelial tumor samples. The interaction scores for the 38 samples are shown in figure 13.

Example 3. Sample preparation, imaging, and imaging analysis of tissue samples with cells expressing PD-L1 and cells expressing CD80

Sample preparation. Formalin Fixed Paraffin Embedded (FFPE) tissue samples were deparaffinized, rehydrated and subjected to antigen retrieval under elevated temperature conditions. Staining was then performed followed by the following steps. First, using RNAscope @ (advanced cell diagnosis), CTLA-4 expression was detected on tissues using 20 pairs of hybridization probes spanning about 1kb of CTLA-4 mRNA. In situ hybridization was observed with TSA-Cy 3. The sections were washed and then quenched with two washes of fresh 100mM benzoyl hydrazine containing 50mM hydrogen peroxide to quench any residual HRP. Sections were washed again before staining with the mouse anti-CD 80 primary antibody. After sections were washed, incubation was performed with anti-murine HRP secondary antibody. Cleaning the slices, thenThe CD80 staining was detected using TSA-Cy 5 (Perkin Elmer). The sections were then washed twice with fresh 100mM benzoyl hydrazine containing 50mM hydrogen peroxide to quench any residual HRP. Sections were washed again and stained with rabbit anti-CD 3 primary antibody. Sections were washed and then incubated with a mixture of anti-rabbit HRP secondary antibody and 4', 6-diamidino-2-phenylindole (DAPI). Sections were washed and then tested for CD3 staining using TSA-AlexaFluor488 @ (Life Technologies). The sections were washed for the last time, and then coverslips with mounting medium were covered on the sections and dried overnight at room temperature.

A similar imaging and analysis process as in example 1 was performed for DAPI, FITC, Cy3 and Cy5 wavelengths. The expression of CTLA-4 and CD80 was used to develop an enrichment algorithm for acquiring 20x images. The analysis was performed to determine the CTLA-4/CD80 interaction score by measuring the total area of CTLA-4 and CD3 positive cells (in pixels) within the proximity of CD80 positive cells divided by the total area of CD3 positive cells (in pixels) multiplied by a factor of 10000. The results are shown in fig. 20.

Example 4. Sample preparation, imaging, and imaging analysis of tissue samples with CTLA-4 expressing cells and CD80 expressing cells.

A procedure similar to that of example 1 was performed, replacing staining and analysis of PD-L1 and PD-1 with staining and analysis of CTLA-4 and CD 80.

Example 5. Sample preparation, imaging and imaging analysis of tissue samples with cells expressing PD-L2 and cells expressing PD-1.

A procedure similar to that of example 1 was performed, replacing the staining and analysis of PD-L1 with that of PD-L2.

Example 6. Sample preparation, imaging and imaging analysis of tissue samples with CTLA-4 expressing cells and CD86 expressing cells.

A procedure similar to that of example 1 was performed, replacing staining and analysis of PD-L1 and PD-1 with staining and analysis of CTLA-4 and CD 86.

Example 7. Sample preparation, imaging and imaging analysis of tissue samples with LAG-3 expressing cells and HLA-DR expressing cells.

A procedure similar to example 1 was performed, replacing the staining and analysis of PD-L1 and PD-1 with that of LAG-3 and HLA-DR.

Example 8. Sample preparation, imaging and imaging analysis of tissue samples with TIM-3 expressing cells and galectin 9 expressing cells.

A procedure similar to example 1 was performed, replacing the staining and analysis of PD-L1 and PD-1 with staining and analysis of TIM-3 and galectin 9.

Example 9. Sample preparation, imaging and imaging analysis of tissue samples with cells expressing 41BB and cells expressing 4.1 BBL.

A procedure similar to example 1 was performed, replacing the staining and analysis of PD-L1 and PD-1 with that of 41BB and 4.1 BBL.

Example 10. Sample preparation, imaging, and imaging analysis of tissue samples having cells expressing OX40 and cells expressing OX 40L.

A procedure similar to that of example 1 was performed, replacing the staining and analysis of PD-L1 and PD-1 with that of OX40 and OX 40L.

Example 11. Sample preparation, imaging and imaging analysis of tissue samples with cells expressing CD40 and cells expressing CD 40L.

A procedure similar to example 1 was performed to exclude staining and analysis of replacement PD-L1 and PD-1 with staining and analysis of CD40 and CD 40L.

Example 12. Sample preparation, imaging and imaging analysis of tissue samples with ICOS expressing cells and ICOSL expressing cells.

A procedure similar to that of example 1 was performed, replacing the staining and analysis of PD-L1 and PD-1 with that of ICOS and ICOSL.

Example 13. Sample preparation, imaging and imaging analysis of tissue samples with GITR-expressing cells and GITRL-expressing cells.

A procedure similar to example 1 was performed, replacing the staining and analysis of PD-L1 and PD-1 with that of GITR and GITRL.

Example 14. Sample preparation, imaging and imaging analysis of tissue samples with cells expressing HLA-DR and cells expressing TCR.

A procedure similar to that of example 1 was performed, replacing staining and analysis of PD-L1 and PD-1 with staining and analysis of HLA-DR and TCR.

Example 15. Sample preparation, imaging, and imaging analysis of tissue samples with cells expressing PD-1, PD-L1, and CD3

A similar process to example 1 was performed, wherein the process was performed without murine anti-S100 antibody. Alternatively, primary and secondary antibodies were removed by microwave after PD-L1 detection. The sections were then stained with rabbit anti-CD 3 primary antibody. Sections were washed and then incubated with a mixture of anti-rabbit HRP secondary antibody and 4', 6-diamidino-2-phenylindole (DAPI). Sections were washed and then detected for CD3 staining using TSA-AlexaFluor488 (Life Technologies). Imaging and analysis was performed similarly to example 1, where spatial proximity (e.g., interaction score) was calculated by dividing the area of PD-1 positive cells in the positive region of PD-L1 (in pixels) by the area of all nucleated cells (in pixels) multiplied by a factor of 10000. The interaction scores for the 29 samples are shown in figure 21.

Paragraph a. A method of scoring a sample comprising tumor tissue taken from a cancer patient, comprising:

(i) determining a score representing spatial proximity between at least one pair of cells using a sample comprising tumor tissue taken from a cancer patient, a first member of the at least one pair of cells expressing a first biomarker and a second member of the at least one pair of cells expressing a second biomarker different from the first biomarker; and

(ii) a score is recorded that, when compared to a threshold, indicates the likelihood that the cancer patient will respond positively to the immunotherapy.

Paragraph B. The method of paragraph a, wherein the first member of the at least one pair of cells comprises a tumor cell and the second member of the at least one pair of cells comprises a non-tumor cell.

Paragraph C. The method of paragraph a or paragraph B, wherein the non-tumor cells comprise immune cells.

Paragraph D. The method of paragraph a, wherein the first member and the second member of the at least one pair of cells comprise immune cells.

Paragraph E. The method of paragraph a, wherein a first member of the at least one pair of cells comprises a tumor cell, a bone marrow cell, or a stromal cell and a second member of the at least one pair of cells comprises an immune cell.

Paragraph F. The method of paragraph E, wherein the tumor cells, bone marrow cells, or stromal cells express PD-L1 and the immune cells express PD-1.

Paragraph G. The method of any of paragraphs a-F, wherein the spatial proximity is evaluated on a pixel scale.

Paragraph H. The method of any of paragraphs a-G, wherein the spatial proximity between at least one pair of cells is in the range of about 1 pixel to about 100 pixels.

Paragraph I. The method of any of paragraphs a-F, wherein the spatial proximity between at least one pair of cells is in the range of about 0.5 μ ι η to about 50 μ ι η.

Paragraph J. The method of paragraph a, the determining step further comprising:

(i) selecting a predetermined number of fields of view obtainable from a sample comprising tumor tissue stained with a plurality of fluorescent markers taken from a cancer patient, the selection biased toward selecting fields of view comprising a greater number of cells expressing a first biomarker than other fields of view;

(ii) for each selected field of view, enlarging the fluorescence signal attributable to the first biomarker by a margin sufficient to encompass proximally located cells expressing the second biomarker; and

(iii) the spatial proximity score is achieved by dividing the first total area of all cells in each selected field of view that express the second biomarker and are encompassed within the expanded fluorescent signal attributable to the cells expressing the first biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor.

Paragraph K. The method of paragraph J, wherein each of the fluorescent labels is for a specific biomarker.

Paragraph L. The method of paragraph J or paragraph K, wherein the plurality of fluorescent labels comprises a first fluorescent label for a first biomarker and a second fluorescent label for a second biomarker.

Paragraph M. The method of any of paragraphs J-L, wherein the margin is in a range of about 1 pixel to about 100 pixels.

Paragraph N. The method of any of paragraphs J-M, wherein the proximally located cells expressing the second biomarker are within about 0.5 to about 50 μ ι η of the plasma membrane of the cells expressing the first biomarker.

Paragraph O. The method of any of paragraphs J-N, wherein the first total area is measured in pixels.

Paragraph P. The method of any of paragraphs J-O, wherein the normalization factor is a second total area of all non-tumor cells in each selected field of view.

Paragraph Q. The method of any of paragraphs J-O, wherein the normalization factor is a second total area of all cells with the ability to express the second biomarker per selected field of view.

Paragraph R. The method of paragraph P or paragraph Q, wherein the second total area is measured in units of pixels.

Paragraph S. The method according to any of paragraphs J-R, wherein the predetermined factor is 10⁴。

Paragraph T. The method of paragraph a, wherein the first member of the at least one pair of cells expresses a first biomarker selected from the group consisting of PD-L1, PD-L2, B7-H3, B7-H4, HLA-DR, galectin 9, CD80, CD86, 4.1BBL, ICOSL, CD40, OX40L, IDO-1, GITRL, and combinations thereof, and the second member of the at least one pair of cells expresses a second biomarker selected from the group consisting of PD-1, TIM3, LAG3, 41BB, OX40, CTLA-4, CD40L, CD28, GITR, ICOS, CD28, and combinations thereof.

Paragraph U. The method of paragraph T, wherein the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses PD-1.

Paragraph V. The method of paragraph T, wherein the first member of the at least one pair of cells expresses PD-L1 and the second member of the at least one pair of cells expresses CD 80.

Paragraph W. The method of paragraph T, wherein the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 80.

Paragraph X. The method of paragraph T, wherein the first member of the at least one pair of cells expresses PD-L2 and the second member of the at least one pair of cells expresses PD-1.

Paragraph Y. The method of paragraph T, wherein the first member of the at least one pair of cells expresses CTLA-4 and the second member of the at least one pair of cells expresses CD 86.

Paragraph Z. The method of paragraph T, wherein a first member of the at least one pair of cells expresses LAG-3 and a second member of the at least one pair of cells expresses HLA-DR.

Paragraph AA. The method of paragraph T, wherein the first member of the at least one pair of cells expresses TIM-3 and the second member of the at least one pair of cells expresses galectin 9.

Paragraph AB. The method of paragraph T, wherein the first member of the at least one pair of cells expresses 41BB and the second member of the at least one pair of cells expresses 4.1 BBL.

Paragraph AC. The method of paragraph T, wherein the first member of the at least one pair of cells expresses OX40 and the second member of the at least one pair of cells expresses OX 40L.

Paragraph AD. The method of paragraph T, wherein the first member of the at least one pair of cells expresses CD40 and the second member of the at least one pair of cells expresses CD 40L.

Paragraph AE. The method of paragraph T, wherein the first member of the at least one pair of cells expresses ICOS and the second member of the at least one pair of cells expresses ICOSL.

Paragraph AF. The method of paragraph T, wherein the first member of the at least one pair of cells expresses GITR and the second member of the at least one pair of cells expresses GITRL.

Paragraph AG. The method of paragraph T, wherein a first member of the at least one pair of cells expresses HLA-DR and a second member of the at least one pair of cells expresses TCR.

Paragraph AH. The method of any of paragraphs a-AG, wherein the threshold value ranges from about 500 to about 5000.

Paragraph AI. A method according to paragraph AH, wherein the threshold is about 900 plus or minus 100.

Paragraph AJ. The method of any of paragraphs a-AI, wherein the immunotherapy comprises immune checkpoint therapy.

Paragraph AK. A method of determining a score representing a spatial proximity between at least one pair of cells selected from a plurality of cells present in a predetermined number of fields of view obtainable from a sample comprising tumor tissue taken from a cancer patient, the method comprising:

(ii) for each selected field of view, enlarging the fluorescence signal attributable to the first specific biomarker by a margin sufficient to encompass proximally located cells expressing the second specific biomarker; and

(iii) the spatial proximity score is achieved by dividing the first total area of all cells in each selected field of view that express the second specific biomarker and are encompassed within the expanded fluorescent signal attributable to the cells expressing the first specific biomarker by a normalization factor, and multiplying the resulting quotient by a predetermined factor.

Paragraph AL. The method of paragraph AK, wherein each of the fluorescent labels is for a specific biomarker.

Paragraph AM. The method of paragraph AK or paragraph AL, wherein the plurality of fluorescent labels comprises a first fluorescent label for the first biomarker and a second fluorescent label for the second biomarker.

Paragraph AN. The method of paragraph AK-paragraph AM, wherein the one or more fluorescent labels comprise a fluorophore conjugated to an antibody or another antibody having binding affinity for a particular biomarker.

Paragraph AO. The method of paragraph AK-paragraph AM, wherein each fluorescent marker comprises a fluorophore selected independently from one or more of the group consisting of DAPI, Cy2, Cy3, 5, Cy5, FITC, TRITC, 488 dye, 555 dye, 594 dye, and Texas Red.

Paragraph AP. The method of paragraph AK-paragraph AO wherein the margin is in the range of about 1 pixel to about 100 pixels.

Paragraph AQ. The method of any of paragraphs AK-paragraphs AP, wherein the proximally located cells expressing the second specific biomarker are within about 0.5 to about 50 μ ι η of the plasma membrane of the cells expressing the first specific biomarker.

Paragraph AR. The method of any of paragraphs AK-paragraphs AQ, wherein the first total area is measured in pixels.

Paragraph AS. The method of any of paragraphs AK-AP, wherein the normalization factor is a second total area of all non-tumor cells in each selected field of view.

Paragraph AT. The method of any of paragraphs AK-paragraphs AP, wherein the normalization factor is a second total area of all cells with the ability to express a second specific biomarker per selected field of view.

A paragraph AU. The method according to paragraph AS or paragraph AT, wherein the second total area is measured in units of pixels.

Paragraph AV. The method of any of paragraphs AK-paragraphs AU, where the predetermined factor is 10⁴。

Paragraph AW. The method of paragraph AK, wherein the Spatial Proximity Score (SPS) is determined by the equation:

Paragraph AX. The method of paragraph AK wherein the first specific biomarker comprises tumor and non-tumor markers and the second specific biomarker comprises non-tumor markers.

Paragraph AY. The method of paragraph a, wherein the method provides superior predictive power compared to quantitatively estimating expression of the first biomarker or quantitatively estimating expression of the second biomarker.

Paragraph AZ. The method according to paragraph AK, wherein the method provides an excellent predictive power compared to a quantitative estimation of the expression of a first specific biomarker or a quantitative estimation of the expression of a second specific biomarker.

Paragraph BA. The method of paragraph AY or paragraph AZ, wherein the predictive power is quantified as a positive predictive value, a negative predictive value, or a combination thereof.

Paragraph BB. The method of paragraph BA, wherein the positive predictive value is 65% or greater.

Paragraph BC. The method of paragraph BB, wherein the positive predictive value is 70% or greater.

Paragraph BD. The method of paragraph BC, wherein the positive predictive value is 75% or greater.

Paragraph BE. The method of paragraph BA, wherein the negative predictive value is 65% or greater.

Paragraph BF. The method of paragraph BE, wherein the negative predictive value is 80% or greater.

While particular embodiments have been shown and described, it will be appreciated that modifications and changes may be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the appended claims.

The exemplary described embodiments herein may be suitably practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed in an extensible and non-limiting sense. Additionally, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. In addition, the phrase "consisting essentially of … …" will be interpreted to include those elements specifically recited as well as additional elements that do not materially affect the basic and novel characteristics of the presently claimed technology. The phrase "consisting of … …" excludes any element not specifically recited.

The present disclosure is not limited in terms of the particular embodiments described in this application. It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope thereof. Functionally equivalent methods and compositions within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The disclosure is to be limited only by the aspects of the following claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compound compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Further, where a feature or aspect of the disclosure is described in terms of a Markush (Markush) group, it will be apparent to one skilled in the art that the disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group.

It will be apparent to those skilled in the art that all ranges disclosed herein, useful for any and all purposes, especially in providing written description, also include any and all possible subranges and combinations of subranges thereof. Any listed range can be readily considered sufficient to describe and achieve the same range divided into at least two, three, four, five, ten, etc. As a non-limiting example, each range disclosed herein may be readily divided into a lower third, a middle third, and an upper third. As will also be understood by those of skill in the art, all languages such as "up to," "at least," "greater than," "less than," and the like include the recited number and refer to ranges that may be subsequently separated into sub-ranges as described above. Finally, as will be understood by those of skill in the art, a range includes each individual member.

All publications, patent applications, and issued patents and other documents cited in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent or other document were specifically and individually indicated to be incorporated by reference in its entirety. In the event that a definition of the present disclosure conflicts with a definition contained in the text incorporated by reference, the present disclosure controls.

Other embodiments are given in the dependent claims.

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Method of scoring a sample comprising tumor tissue

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