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Measurement of Immune Infiltration in ER, PR, HER2 IHC Subtypes Reveals Different Populations That May Benefit from Immunotherapy Pinky Tripathi 1 , Nam Tran 1 , Raghavkrishna Padmanabhan 1 , Richard Hartsfield 1 , Edward J. Moler 1 , Nicholas Hoe 1 , Kenneth Bloom 2 1 Clarient Diagnostic Services, Inc., Aliso Viejo, California, 2 Clarient Pathology Services, Aliso Viejo, California. Background Results: Integrated Analysis of IHC4 and Immune Markers Methods Discussion Expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as measured by immunohistochemistry, is routinely used to guide appropriate therapy in breast cancer. HER2 targeted therapy is used to treat HER2 overexpressing patients and endocrine therapy is used to treat ER + or PR + patients. However, therapeutic options are limited for patients who are triple negative, relapsed HER2 overexpressed patients, and ER + or PR + patients who are refractory to endocrine therapy. For these breast cancer patients, immunotherapy has the potential to improve survival by targeting cancer cells. Although, the role of immune response in breast cancer is not fully understood, studies have observed high number of natural killer (NK) cells, B cells, and cytotoxic T cells suppress tumor growth while high number of macrophages, and regulatory T cells (Treg) promote tumor growth. The purpose of this study is to measure levels of immune cells infiltration (cytotoxic, helper, and regulatory T cells, NK cells, and macrophages) between different ER, PR, and HER2 IHC subtypes. Combined IHC4 and immune markers were profiled from a single FFPE slide using a hyperplexed MultiOmyx assay. Total of 106 breast tumor TMAs were assessed for immune cells infiltration in each of the following IHC4 subtypes: ER + /PR + /HER2 - , ER + /PR + /HER2 + , ER + /PR - /HER2 - , ER + /PR - /HER2 overexpressed , ER - /PR - /HER2 overexpressed , and triple negative. Immune cells infiltration is defined as a percentage of positive immune cells relative to the total number of stromal cells. PanCK was used to differentiate between tumor and stromal regions. ER - /PR - /HER2 overexpressed subtype displayed the highest percentage of not only CD4, CD8 positive cells, but also Ki67 positive cells, followed by the triple negatives, and ER + /PR - /HER2 overexpressed . In the remaining three subtypes, the percentage of CD4, and CD8 positive cells are 1-2% (Fig 2C). Percentage of CD56 positive natural killer cells remained consistent across all subtypes (data not shown). Co-expression analysis (Fig 2D) in ER - /PR - /HER2 overexpressed illustrates, despite high percentage of CD8+ cytotoxic T lymphocytes, Ki67 positive cells were abundant in the tumor. The data suggests: Presence of high intratumoral T cells in ER - /PR - /HER2 overexpressed does not indicate functionally active T cells Possible evasion of host tumor-immune response by immunoinhibitory factors Potential benefit from immunotherapy targeting immune checkpoints (anti-PD-L1, anti-CTLA-4) Comprehensive immunophenotyping requires identification of specific immune cell types, and differentiation of activated immune cells from inhibitory immune cells. + 2. Stain Slide 3. Acquire immunofluorescence Cy3-Ab A Cy5-Ab B Staining Round 1 Staining Round 2 1. Acquire Background 5. Acquire New Background Staining Round 3 Staining Round (n ) 4. Inactivate Dye 6. Repeat (1…n) New Ab Figure 1: MultiOmyx IF multiplexing scheme from a single tissue section Conjugated fluorescent antibodies were applied to a slide, followed by whole slide imaging. The dye was chemically inactivated, enabling a second round of staining with another fluorescent antibody. The process was performed multiple times from a single slide. Tissue microarrays were constructed from 106 breast cancer patients consisting of 36 triple negatives, 11 HER2 overexpressed (ER - /PR - ), 7 ER + /PR - /HER2 - , 24 ER + /PR + /HER2 - , 8 ER + /PR - /HER2 + , and 20 ER + /PR + /HER2 + assessed by a pathologist based on immunohistochemical stains. MultiOmyx TM hyperplexed immunofluorescence assay was performed on the constructed TMAs to profile IHC4 (HER2, ER, PR, Ki67), and immune (CD3, CD4, CD8, CD45RO, CD56, CD68, FOXP3) markers. Tumor Stroma CD4 CD8 CD68 25.3 74.7 ER + PR + HER2 - 68.7 49.9 4.6 ER PR HER2 Ki67 Ki67 ER + PR + HER2 + 20.0 80.0 90.2 88.3 88.0 8.0 Tumor Stroma CD4 CD8 CD68 ER PR HER2 Ki67 Ki67 25.4 74.6 ER + PR - HER2 - 64.2 7.5 Tumor Stroma CD4 CD8 CD68 ER PR HER2 Ki67 Ki67 15.9 84.1 68.3 62.6 ER + PR - HER2 overexpressed 4.9 2.4 18.3 Tumor Stroma CD4 CD8 CD68 ER PR HER2 Ki67 Ki67 ER - PR - HER2 overexpressed 2.7 97.3 81.3 23.4 4.8 21.5 56.9 13.2 Tumor Stroma CD4 CD8 CD68 ER PR HER2 Ki67 Ki67 ER - PR - HER2 - 2.7 97.3 81.3 8.2 4.8 2.0 66.5 22.2 Tumor Stroma CD4 CD8 CD68 ER PR HER2 Ki67 Ki67 IHC Subtypes (n=106) Tumor (% + cells) Stroma (% + cells) Total (% / mm 2 ) ER PR HER2 CD4 CD8 CD68 Ki67 ER+PR+HER2- (24) 75.8 67.5 - 1.1 <1 1.3 2.7 ER+PR+HER2+ (20) 79.9 57.2 81.7 2.4 <1 2.1 2.3 ER+PR-HER2- (07) 85.6 5.4 - 2.3 1.0 1.3 4.6 ER+PR-HER2OverExp(08) 70.7 11.2 79.4 3.5 <1 2.1 4.3 ER-PR-HER2OverExp (11) - 1.9 78.4 9.2 4.1 3.9 9.3 Triple Negatives (36) - 1.2 - 5.8 1.2 3.9 5.6 Figure 2: A,B. Representative multiplexed IF color blended images across six IHC4 subtypes for ER,PR, HER2, Ki67 (top row), CD4, CD8, CD68, and CK (bottom row). 2 sets of representative images are shown. C. Classification and quantitation of cells positive for IHC4 and immune biomarkers across all 106 samples. ER, PR, HER2 is reported as percentage of positive cells in tumor. CD4, CD8, CD68 is reported as a percentage of positive cells in stroma. Ki67 is reported as a percentage of Ki67 positive cells/mm 2 (total cells). D. Co-expression analysis of ER, PR, HER2, and Ki67 in tumor and CD4, CD8, CD68, and Ki67 in stroma. Each marker is calculated as percentage of cells positive in their respective regions. Matching color blended images are shown in figure 2A above. ER PR HER2 Ki67 ER + PR + HER2 - CD4 CD8 CD68 CK ER + PR + HER2 + ER + PR - HER2 overexpressed ER - PR - HER2 overexpressed S15010017a_P036 Triple Negatives ER PR HER2 Ki67 CD4 CD8 CD68 CK A. B. C. D. 0 5 10 15 20 % Expression in Stroma CD4 CD8 CD68 0 25 50 75 100 % Expression in Tumor ER PR HER2 ER + PR - HER2 -S15010017a_P036 © 2015 General Electric Company. GE, the GE Monogram, and MultiOmyx are trademarks of General Electric Company. Clarient Diagnostic Services, Inc. is a CLIA-licensed laboratory and a division of General Electric Company. September 2015 JB33907US
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AACR Immune Infiltration In ER, PR, HER2 IHC Subtypes

Feb 14, 2017

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Page 1: AACR Immune Infiltration In ER, PR, HER2 IHC Subtypes

Measurement of Immune Infiltration in ER, PR, HER2 IHC Subtypes Reveals Different Populations That May Benefit from Immunotherapy

Pinky Tripathi1, Nam Tran1, Raghavkrishna Padmanabhan1, Richard Hartsfield1, Edward J. Moler1, Nicholas Hoe1, Kenneth Bloom2 1Clarient Diagnostic Services, Inc., Aliso Viejo, California, 2Clarient Pathology Services, Aliso Viejo, California.

Background Results: Integrated Analysis of IHC4 and Immune Markers

Methods

Discussion

Expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as measured by immunohistochemistry, is routinely used to guide appropriate therapy in breast cancer. HER2 targeted therapy is used to treat HER2 overexpressing patients and endocrine therapy is used to treat ER+ or PR+ patients. However, therapeutic options are limited for patients who are triple negative, relapsed HER2 overexpressed patients, and ER+ or PR+ patients who are refractory to endocrine therapy. For these breast cancer patients, immunotherapy has the potential to improve survival by targeting cancer cells. Although, the role of immune response in breast cancer is not fully understood, studies have observed high number of natural killer (NK) cells, B cells, and cytotoxic T cells suppress tumor growth while high number of macrophages, and regulatory T cells (Treg) promote tumor growth. The purpose of this study is to measure levels of immune cells infiltration (cytotoxic, helper, and regulatory T cells, NK cells, and macrophages) between different ER, PR, and HER2 IHC subtypes.

Combined IHC4 and immune markers were profiled from a single FFPE slide using a hyperplexed MultiOmyx assay. Total of 106 breast tumor TMAs were assessed for immune cells infiltration in each of the following IHC4 subtypes: ER+/PR+/HER2-, ER+/PR+/HER2+, ER+/PR-/HER2-, ER+/PR-/HER2overexpressed, ER-/PR-/HER2overexpressed, and triple negative. Immune cells infiltration is defined as a percentage of positive immune cells relative to the total number of stromal cells. PanCK was used to differentiate between tumor and stromal regions. ER-/PR-/HER2overexpressed subtype displayed the highest percentage of not only CD4, CD8 positive cells, but also Ki67 positive cells, followed by the triple negatives, and ER+/PR-/HER2overexpressed. In the remaining three subtypes, the percentage of CD4, and CD8 positive cells are 1-2% (Fig 2C). Percentage of CD56 positive natural killer cells remained consistent across all subtypes (data not shown). Co-expression analysis (Fig 2D) in ER-/PR-/HER2overexpressed illustrates, despite high percentage of CD8+ cytotoxic T lymphocytes, Ki67 positive cells were abundant in the tumor. The data suggests:

• Presence of high intratumoral T cells in ER-/PR-/HER2 overexpressed does not indicate functionally active T cells • Possible evasion of host tumor-immune response by immunoinhibitory factors • Potential benefit from immunotherapy targeting immune checkpoints (anti-PD-L1, anti-CTLA-4) Comprehensive immunophenotyping requires identification of specific immune cell types, and differentiation of activated immune cells from inhibitory immune cells.

+

2. Stain Slide 3. Acquire immunofluorescence

Cy3-Ab A Cy5-Ab B Staining Round 1 Staining Round 2

1. Acquire Background

5. Acquire New Background

Staining Round 3

Staining Round (n )

4. Inactivate Dye 6. Repeat (1…n) New Ab

Figure 1: MultiOmyx IF multiplexing scheme from a single tissue section Conjugated fluorescent antibodies were applied to a slide, followed by whole slide imaging. The dye was chemically inactivated, enabling a second round of staining with another fluorescent antibody. The process was performed multiple times from a single slide.

Tissue microarrays were constructed from 106 breast cancer patients consisting of 36 triple negatives, 11 HER2 overexpressed (ER-/PR-), 7 ER+/PR-/HER2-, 24 ER+/PR+/HER2-, 8 ER+/PR-/HER2+, and 20 ER+/PR+/HER2+ assessed by a pathologist based on immunohistochemical stains. MultiOmyxTM hyperplexed immunofluorescence assay was performed on the constructed TMAs to profile IHC4 (HER2, ER, PR, Ki67), and immune (CD3, CD4, CD8, CD45RO, CD56, CD68, FOXP3) markers.

Tumor Stroma

CD4 CD8

CD68

25.3 74.7

ER+PR+HER2-

68.7

49.9

4.6

ER PR

HER2 Ki67 Ki67

ER+PR+HER2+

20.0 80.0

90.2

88.3

88.0

8.0

Tumor Stroma

CD4 CD8

CD68 ER PR

HER2 Ki67 Ki67

25.4 74.6

ER+PR-HER2-

64.2

7.5

Tumor Stroma

CD4 CD8

CD68 ER PR

HER2 Ki67 Ki67

15.9 84.1

68.3

62.6

ER+PR-HER2overexpressed

4.9

2.4

18.3

Tumor Stroma

CD4 CD8

CD68 ER PR

HER2 Ki67 Ki67

ER-PR-HER2overexpressed

2.7 97.3

81.3 23.4

4.8

21.5

56.9 13.2

Tumor Stroma

CD4 CD8

CD68 ER PR

HER2 Ki67 Ki67

ER-PR-HER2-

2.7 97.3

81.3

8.2

4.8 2.0

66.5 22.2

Tumor Stroma

CD4 CD8

CD68 ER PR

HER2 Ki67 Ki67

IHC Subtypes (n=106) Tumor (% + cells) Stroma (% + cells) Total

(% / mm2)

ER PR HER2 CD4 CD8 CD68 Ki67

ER+PR+HER2- (24) 75.8 67.5 - 1.1 <1 1.3 2.7

ER+PR+HER2+ (20) 79.9 57.2 81.7 2.4 <1 2.1 2.3

ER+PR-HER2- (07) 85.6 5.4 - 2.3 1.0 1.3 4.6

ER+PR-HER2OverExp(08) 70.7 11.2 79.4 3.5 <1 2.1 4.3

ER-PR-HER2OverExp (11) - 1.9 78.4 9.2 4.1 3.9 9.3

Triple Negatives (36) - 1.2 - 5.8 1.2 3.9 5.6

Figure 2: A,B. Representative multiplexed IF color blended images across six IHC4 subtypes for ER,PR, HER2, Ki67 (top row), CD4, CD8, CD68, and CK (bottom row). 2 sets of representative images are shown.

C. Classification and quantitation of cells positive for IHC4 and immune biomarkers across all 106 samples. ER, PR, HER2 is reported as percentage of positive cells in tumor. CD4, CD8, CD68 is reported as a percentage of positive cells in stroma. Ki67 is reported as a percentage of Ki67 positive cells/mm2 (total cells).

D. Co-expression analysis of ER, PR, HER2, and Ki67 in tumor and CD4, CD8, CD68, and Ki67 in stroma. Each marker is calculated as percentage of cells positive in their respective regions. Matching color blended images are shown in figure 2A above.

ER P

R H

ER2

Ki67

ER+PR+HER2-

CD4

CD

8 CD

68 C

K

ER+PR+HER2+ ER+PR-HER2overexpressed ER-PR-HER2overexpressed S15010017a_P036 Triple Negatives

ER P

R H

ER2

Ki67

CD

4 C

D8

CD68

CK

A.

B.

C.

D.

0

5

10

15

20% Expression in Stroma CD4

CD8

CD68

0

25

50

75

100% Expression in Tumor

ER

PR

HER2

ER+PR-HER2-S15010017a_P036

© 2015 General Electric Company. GE, the GE Monogram, and MultiOmyx are trademarks of General Electric Company. Clarient Diagnostic Services, Inc. is a CLIA-licensed laboratory and a division of General Electric Company.

September 2015 JB33907US