s i Quantification of Rare PD-L1 and Other Immunology ... of Rare PD-L1 and Other Immunology...
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Quantification of Rare PD-L1 and Other Immunology Biomarker Expressing Leukocytes and CTCs in Peripheral Blood of Cancer Patients Adam Jendrisak, Nadia Ebrahim, Angel Rodriguez, Rachel Krupa, David Lu, Mahipal Suraneni, Jiyun Byun, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore Epic Sciences, Inc., San Diego, California Background • Expression of PD-L1 on tumor tissue and immune cell markers (CD3, CD4, CD8, etc.) on tumor infiltrating lymphocytes (TILs) are often associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, PD-L1 diagnostic tests suffer from high co-morbidities and from significant false positives and false negatives. • Expression of PD-1 and Tim-3 on TILs is associated with immune exhaustion. • Utilizing a non-invasive liquid biopsy, we sought to examine the expression of checkpoint markers (PD-L1, Tim-3) and immune cell markers (CD3, CD8) on circulating tumor cells (CTCs) and leukocyte cell populations for the purpose of developing an improved predictive and pharmacodynamic biomarker for approved PD- 1/PD-L1 checkpoint inhibitors and novel immunotherapy drugs in development. PD - L1 Expression in CTC and Leukocytes of Lung Ca PD-L1 Protein Assay Development www.epicsciences.com • Epic Sciences’ platform has low limit of detection, ability to archive patient blood samples and ability to quantify biomarker expression on both CTCs and leukocytes simultaneously. • Detection of leukocyte subtypes such as CD3, CD8, CD14 and CD56 cells, will allow us to further characterize PD-L1 and Tim-3 in T-lymphocytes and other immune cell types. • Development of a liquid biopsy-based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leukocytes will allow real time assessment of response to immune checkpoints inhibitors. Tim - 3 Expression in Healthy Donor Leukocytes Conclusions • Blood samples were drawn from 3 healthy donors, 18 non-cancerous lung disease, 33 lung cancer, and 1 bladder cancer patients and sent to Epic Sciences for processing with Epic Sciences’ immunoassays. • Panels of immunoassays include staining of checkpoint markers (PD-L1, Tim-3) and immune cell markers (CD3, CD8). Methods Schematic of Epic CTC platform CTC enumeration, morphology and protein analyses workflow: 1) Nucleated cells from one 10mL blood draw were plated onto 10-12 slides with each slide having ~3 million cells 2) Slides stained with DAPI and a combination of two or three other markers, including cytokeratin (CK), CD3, CD8, CD45, PD-L1 and Tim-3 3) Slides scanned 4) All cells measured by a multi-parametric digital pathology algorithm for morphological features and protein expression 5) Biomarker analyses were performed on both CTCs and leukocytes Percentage of PD - L1(+) Leukocytes in 54 Sample Cohort CD3 Expression in Jurkat Cells and Healthy Donor Leukocytes CK(+)/ CD45(-)/ PD-L1(+) CK(-)/ CD45(-)/ PD-L1(+) CK(-)/ CD45(+)/ PD-L1(+) 4) SINGLE CELL DIGITAL PATHOLOGY 1) SLIDE PREPARATION 2) CELL STAINING 3) SCANNING Nuclear Features Cytoplasmic Features Cell Features Nuclear Area Cytoplasmic Area AR Expression Nuc. Convex Area Cyto Convex Area CK Expression Nuc Major Axis Cyto Major Axis N/C Ratio Nuclear Minor Axis Cyto Minor Axis Nuclear Circularity Cyto Circularity Nuclear Solidity Cyto Solidity Nuclear Entropy Cyto Entropy Nuclear Speckling Cyto Speckling Nucleoli Presence Cyto Presence Circulating tumor cells (CTCs) Immune cells Epithelial (CK+) CTCs Small CTC CK- CTC CTC Cluster 5) BIOMARKER ANALYSIS ON CTC AND LEUKOCYTES Immune Panel • PD-L1 • Tim-3 • CD3 • CD4 • CD8 • CD14 • CD56 CK, CD45, DAPI, PD-L1, etc. Figure 2: Demonstration of PD-L1 assay specificity: (A) PD-L1-specific antibody and species-matched isotype control were tested in negative (Colo205) and high (H820) PD-L1-expressing cell lines. No specific staining was seen in negative control cell lines or with isotype control antibody. (B) Interferon (IFN)-gamma treatment increases PD-L1 expression in Colo205 and A549 cell line cells. PD-L1 expression in IFN-gamma-treated SU-DHL-1 cells remain unchanged, likely due to the up-regulation of cytokine signaling suppressor genes in this particular cell line. PD-L1 expression in cell lines: +/- interferon-gamma treatment PD-L1 expression (log2 scale) - + - + - + 1 2 4 8 16 32 64 128 256 512 Colo205 A549 SU-DHL-1 IFN-gamma: Colo205 isotype Colo205 PD-L1 H820 isotype H820 PD-L1 1 2 4 8 16 32 64 128 256 512 PD-L1 antibody and isotype controls PD-L1 expression (log2 scale) CK Morphology CD45 Composite DAPI CD45 Tim-3 Composite DAPI CD45 Tim-3 Jurkat Cells (Positive Control) Healthy Donor Leukocytes Composite DAPI CD3 CD8, CD45 and PD - L1 Co - Expression in Bladder Ca Leukocytes CD8(-)/CD45(-)/PD-L1(+) CD8(+)/CD45(+)/PD-L1(-) Healthy Donor Leukocytes Healthy Donor Leukocytes Composite DAPI CD3 composite DAPI CK CD45 PD-L1 composite DAPI CK CD45 PD-L1 composite DAPI CK CD45 PD-L1 Composite DAPI CD8 CD45 PD-L1 Composite DAPI CD8 CD45 PD-L1 Percent of Cells (%) Minimum Maximum Median Mean Healthy Donor 0.0076% 0.0166% 0.0077% 0.0106% Lung Disease (non-Cancerous) 0.0000% 0.0510% 0.0000% 0.0067% Lung Cancer 0.0000% 0.1378% 0.0118% 0.0213%
Transcript of s i Quantification of Rare PD-L1 and Other Immunology ... of Rare PD-L1 and Other Immunology...
Quantification of Rare PD-L1 and Other Immunology Biomarker Expressing Leukocytes
and CTCs in Peripheral Blood of Cancer PatientsAdam Jendrisak, Nadia Ebrahim, Angel Rodriguez, Rachel Krupa, David Lu, Mahipal Suraneni, Jiyun Byun, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore
Epic Sciences, Inc., San Diego, California
Background
• Expression of PD-L1 on tumor tissue and immune cell markers (CD3, CD4, CD8, etc.) on tumor infiltrating lymphocytes (TILs) are often associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, PD-L1 diagnostic tests suffer from high co-morbidities and from significant false positives and false negatives.
• Expression of PD-1 and Tim-3 on TILs is associated with immune exhaustion. • Utilizing a non-invasive liquid biopsy, we sought to examine the expression of checkpoint markers (PD-L1,
Tim-3) and immune cell markers (CD3, CD8) on circulating tumor cells (CTCs) and leukocyte cell populations for the purpose of developing an improved predictive and pharmacodynamic biomarker for approved PD-1/PD-L1 checkpoint inhibitors and novel immunotherapy drugs in development.
PD-L1 Expression in CTC and Leukocytes of Lung Ca
PD-L1 Protein Assay Development
www.epicsciences.com
• Epic Sciences’ platform has low limit of detection, ability to archive patient blood samples and ability toquantify biomarker expression on both CTCs and leukocytes simultaneously.
• Detection of leukocyte subtypes such as CD3, CD8, CD14 and CD56 cells, will allow us to further characterizePD-L1 and Tim-3 in T-lymphocytes and other immune cell types.
• Development of a liquid biopsy-based platform that is capable of simultaneously measuring immunebiomarkers in CTCs as well as leukocytes will allow real time assessment of response to immunecheckpoints inhibitors.
Tim-3 Expression in Healthy Donor Leukocytes
Conclusions
• Blood samples were drawn from 3 healthy donors, 18 non-cancerous lung disease, 33 lung cancer, and 1 bladder cancerpatients and sent to Epic Sciences for processing with Epic Sciences’ immunoassays.
• Panels of immunoassays include staining of checkpoint markers (PD-L1, Tim-3) and immune cell markers (CD3, CD8).
Methods
Schematic of Epic CTC platform CTC enumeration, morphology and protein analyses workflow:1) Nucleated cells from one 10mL blood draw were plated onto 10-12 slides with each slide having ~3 million cells2) Slides stained with DAPI and a combination of two or three other markers, including cytokeratin (CK), CD3, CD8, CD45, PD-L1
and Tim-33) Slides scanned4) All cells measured by a multi-parametric digital pathology algorithm for morphological features and protein expression5) Biomarker analyses were performed on both CTCs and leukocytes
Percentage of PD-L1(+) Leukocytes in 54 Sample Cohort
CD3 Expression in Jurkat Cells and Healthy Donor Leukocytes
CK(+)/CD45(-)/PD-L1(+)
CK(-)/CD45(-)/PD-L1(+)
CK(-)/CD45(+)/PD-L1(+)
4) SINGLE CELL DIGITAL PATHOLOGY
1) SLIDE PREPARATION 2) CELL STAINING 3) SCANNING
Nuclear Features Cytoplasmic Features Cell Features
Nuclear Area Cytoplasmic Area AR Expression
Nuc. Convex Area Cyto Convex Area CK Expression
Nuc Major Axis Cyto Major Axis N/C Ratio
Nuclear Minor Axis Cyto Minor Axis
Nuclear Circularity Cyto Circularity
Nuclear Solidity Cyto Solidity
Nuclear Entropy Cyto Entropy
Nuclear Speckling Cyto Speckling
Nucleoli Presence Cyto Presence
Circulating tumor cells (CTCs)
Immune cells
Epithelial (CK+) CTCs
Small CTC CK- CTC CTC Cluster
5) BIOMARKER ANALYSIS ON CTC AND LEUKOCYTES
Immune Panel
• PD-L1
• Tim-3
• CD3
• CD4
• CD8
• CD14
• CD56
CK, CD45, DAPI, PD-L1, etc.
Figure 2: Demonstration of PD-L1 assay specificity: (A) PD-L1-specific antibody and species-matched isotype control were testedin negative (Colo205) and high (H820) PD-L1-expressing cell lines. No specific staining was seen in negative control cell lines orwith isotype control antibody. (B) Interferon (IFN)-gamma treatment increases PD-L1 expression in Colo205 and A549 cell linecells. PD-L1 expression in IFN-gamma-treated SU-DHL-1 cells remain unchanged, likely due to the up-regulation of cytokinesignaling suppressor genes in this particular cell line.
P D -L 1 e x p re s s io n in c e ll lin e s :
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P D -L 1 a n t ib o d y a n d is o ty p e c o n tro ls
PD
-L1
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(lo
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CK Morphology
CD45
Composite DAPI CD45 Tim-3
Composite DAPI CD45 Tim-3
Jurkat Cells(Positive Control)
Healthy DonorLeukocytes
Composite DAPI CD3
CD8, CD45 and PD-L1 Co-Expression in Bladder Ca Leukocytes
CD8(-)/CD45(-)/PD-L1(+)
CD8(+)/CD45(+)/PD-L1(-)
Healthy DonorLeukocytes
Healthy DonorLeukocytes
Composite DAPI CD3
composite DAPI CK CD45 PD-L1
composite DAPI CK CD45 PD-L1
composite DAPI CK CD45 PD-L1Composite DAPI CD8 CD45 PD-L1
Composite DAPI CD8 CD45 PD-L1
Percent of Cells (%)
Minimum Maximum Median Mean
Healthy Donor 0.0076% 0.0166% 0.0077% 0.0106%
Lung Disease (non-Cancerous) 0.0000% 0.0510% 0.0000% 0.0067%
Lung Cancer 0.0000% 0.1378% 0.0118% 0.0213%
