Nikita Mani

Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer

PD-L1 is expressed in a percentage of lung cancer patients and those patients show increased likelihood of response to PD-1 axis therapies. However, the methods and assays for the assessment of PD-L1 using immunohistochemistry are variable and PD-L1 expression appears to be highly heterogeneous. Here, we examine assay heterogeneity parameters toward the goal of determining variability of sampling and the variability due to pathologist-based reading of the immunohistochemistry slide. SP142, a rabbit monoclonal antibody, was used to detect PD-L1 by both chromogenic immunohistochemistry and quantitative immunofluorescence using a laboratory-derived test. Five pathologists scored the percentage of PD-L1 positivity in tumor- and stromal-immune cells of 35 resected non-small cell lung cancer cases, each represented on three separate blocks. An intraclass correlation coefficient of 94% agreement was seen among the pathologists for the assessment of PD-L1 in tumor cells, but only 27% agreement was seen in stromal/immune cell PD-L1 expression. The block-to-block reproducibility of each pathologist’s score was 94% for tumor cells and 75% among stromal/immune cells. Lin’s concordance correlation coefficient between pathologists’ readings and the mean immunofluorescence score among blocks was 94% in tumor and 68% in stroma. Pathologists were highly concordant for PD-L1 tumor scoring, but not for stromal/immune cell scoring. Pathologist scores and immunofluorescence scores were concordant for tumor tissue, but not for stromal/immune cells. PD-L1 expression was similar among all the three blocks from each tumor, indicating that staining of one block is enough to represent the entire tumor and that the spatial distribution of heterogeneity of expression of PD-L1 is within the area represented in a single block. Future studies are needed to determine the minimum representative tumor area for PD-L1 assessment for response to therapy.

Loss of antigenicity with tissue age in breast cancer

Archived tumor specimens, particularly those collected by large cooperative groups and trials, provide a wealth of material for post hoc clinical investigation. As these tissues are rigorously collected and preserved for many decades, subsequent use of the specimens to answer clinical questions must rely on the assumption that expression and detection of target biomarkers are not degraded with time. To test this assumption, we measured the expression of estrogen receptor (ER), human epidermal growth receptor 2 (HER2), and Ki67 in human breast carcinoma using quantitative immunofluorescence (QIF) in a series of formalin-fixed paraffin-embedded (FFPE) tissues from 1295 individual patients preserved for 7 to 53 years in four cohorts on tissue microarrays. Protein expression was measured using the automated quantitative analysis method for QIF. Change in quantitative protein expression over time was estimated in positive cases using both Pearson’s correlation and a polynomial regression analysis with a random effects model. The average signal decreased with preservation time for all biomarkers measured. For ER and HER2, there was an average of 10% signal loss after 9.9 years and 8.5 years, respectively, compared with the most recent tissue. Detection of Ki67 expression was lost more rapidly, with 10% signal loss in just 4.5 years. Overall, these results demonstrate the need for adjustment of tissue age when studying FFPE biospecimens. The rate of antigenicity loss is biomarker specific and should be considered as an important variable for studies using archived tissues.

Spatially resolved and quantitative analysis of VISTA/PD-1H as a novel immunotherapy target in human non-small cell lung cancer

Purpose: Determine the localized expression pattern and clinical significance of VISTA/PD-1H in human non–small cell lung cancer (NSCLC). Experimental Design: Using multiplex quantitative immunofluorescence (QIF), we performed localized measurements of VISTA, PD-1, and PD-L1 protein in 758 stage I–IV NSCLCs from 3 independent cohorts represented in tissue microarray format. The targets were selectively measured in cytokeratin+ tumor epithelial cells, CD3+ T cells, CD4+ T-helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes and CD68+ tumor-associated macrophages. We determined the association between the targets, clinicopathological/molecular variables and survival. Genomic analyses of lung cancer cases from TCGA were also performed. Results: VISTA protein was detected in 99% of NSCLCs with a predominant membranous/cytoplasmic staining pattern. Expression in tumor and stromal cells was seen in 21% and 98% of cases, respectively. The levels of VISTA were positively associated with PD-L1, PD-1, CD8+ T cells and CD68+ macrophages. VISTA expression was higher in T-lymphocytes than in macrophages; and in cytotoxic T cells than in T-helper cells. Elevated VISTA was associated with absence of EGFR mutations and lower mutational burden in lung adenocarcinomas. Presence of VISTA in tumor compartment predicted longer 5-year survival. Conclusions: VISTA is frequently expressed in human NSCLC and shows association with increased tumor-infiltrating lymphocytes, PD-1 axis markers, specific genomic alterations and outcome. These results support the immunomodulatory role of VISTA in human NSCLC and suggests its potential as therapeutic target. Clin Cancer Res; 24(7); 1562–73. ©2017 AACR.

A dormant TIL phenotype defines non-small cell lung carcinomas sensitive to immune checkpoint blockers

The biological determinants of sensitivity and resistance to immune checkpoint blockers are not completely understood. To elucidate the role of intratumoral T-cells and their association with the tumor genomic landscape, we perform paired whole exome DNA sequencing and multiplexed quantitative immunofluorescence (QIF) in pre-treatment samples from non-small cell lung carcinoma (NSCLC) patients treated with PD-1 axis blockers. QIF is used to simultaneously measure the level of CD3+ tumor infiltrating lymphocytes (TILs), in situ T-cell proliferation (Ki-67 in CD3) and effector capacity (Granzyme-B in CD3). Elevated mutational load, candidate class-I neoantigens or intratumoral CD3 signal are significantly associated with favorable response to therapy. Additionally, a “dormant” TIL signature is associated with survival benefit in patients treated with immune checkpoint blockers characterized by elevated TILs with low activation and proliferation. We further demonstrate that dormant TILs can be reinvigorated upon PD-1 blockade in a patient-derived xenograft model.Immune checkpoint blockade (ICB) only induces tumour response in a subset of non-small cell lung carcinomas (NSCLC). Here the authors do whole genome sequencing and multiplexed quantitative immunofluorescence on patient samples and identify a “dormant” T-cell signature associated with sensitivity to ICB.

Immune marker profiling and PD-L1 expression across Non-Small Cell Lung Cancer mutations.

Introduction: Programmed death 1/programmed death ligand 1 (PD‐L1) axis inhibitors have been proven effective, especially in patients with tumors expressing PD‐L1. Their clinical efficacy in patients with EGFR‐activating mutations is still unclear, whereas KRAS mutations seem to be associated with good response. Methods: We used multiplexed quantitative immunofluorescence to investigate PD‐L1 expression and to characterize tumor infiltrating lymphocyte (TIL) populations and their activation status in more than 150 NSCLC patients with known mutation status. Results: PD‐L1 expression was significantly lower in EGFR‐mutant compared to KRAS‐mutant, and EGFR/KRAS wild‐type (WT) tumors. KRAS mutant tumors were more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis by TIL activation status revealed that EGFR mutants had a high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present they were almost always active. Additionally, we found differences between EGFR mutation sites in CD8+ expression and the TIL activation profile. Finally, activated EGFR correlated with increased PD‐L1 expression in EGFR mutants but not in EGFR WT, whereas TIL activation was associated with higher PD‐L1 only in EGFR/KRAS WT. Conclusions: Our findings show the unique immune profile of EGFR‐mutant tumors. The high frequency of inactive TILs could explain the low immunotherapy response rates in these patients, whereas PD‐L1 as a predictive biomarker may reflect the constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD‐L1 levels and TILs activation.

Abstract 5600: Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC)

Introduction: The ineffective anti-tumor immune response is characterized by increased immune suppressive signals in the tumor microenvironment. In particular, T-cells recognizing tumor antigens can express diverse immune inhibitory receptors mediating lymphocyte inactivation and limiting tumor rejection. Blockade of these receptors such as PD-1 induces prominent clinical benefit in patients with NSCLC. However, the expression and significance of additional potentially actionable immune inhibitory receptors in lung cancer is poorly understood. Methods: After careful validation of assays and using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (clone EH33, CST), LAG-3 (Clone 17B4, Abcam) and TIM-3 (clone D5D5R, CST) in 698 stages I-IV formalin-fixed paraffin embedded (FFPE) lung carcinomas represented in three tissue microarrays (cohort #1 [Yale n=186], cohort #2 [Yale n=192, and cohort #3 [Greece n=320]). We also included a collection of lung adenocarcinomas with molecular annotation (cohort #4 [Yale n=106]). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers and with major clinico-pathological variables, driver mutations and survival were studied. Results: All the targets were detected predominantly in CD3+ T-cells with membranous staining. Expression of PD-1, LAG-3 and TIM-3 in T-cells across all NSCLC cohorts was 68.7%, 39.7% and 55.8%, respectively. Elevated levels of PD-1, LAG-3 or TIM-3 were significantly associated with increased tumor infiltrating lymphocytes and with the co-expression of one or more of the other inhibitory receptors (P Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in NSCLC, show frequent co-expression and association with elevated CD3+ T-cells. Our results support the biological role of PD-1, LAG-3 and TIM-3 in NSCLC and suggest co-activation of these immune inhibitory pathways in a proportion of cases. Modulation of these receptors could enhance the anti-tumor immune response in lung cancer. Citation Format: Ila J. Datar, Jun Wang, Nikita Mani, Franz Villarroel-Espindola, Patrick Ryan, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, David L. Rimm, Leiping Chen, Roy Herbst, Kurt Schalper. Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5600. doi:10.1158/1538-7445.AM2017-5600

Abstract P5-07-09: Heterogeneity of tumor infiltrating lymphocytes in breast cancer and its impact for use as a biomarker

Background: In breast cancer, elevated tumor infiltrating lymphocytes (TILs) is associated with PD-L1 expression, hormone receptors negativity, and better outcome. The presence of numerous CD8+ cytotoxic T cells in pre-treatment specimens is associated with clinical benefit from PD-1 axis blockade in melanoma and lung cancer, suggesting its predictive value. Despite recent efforts to standardize the pathologist evaluation of TILs in breast cancer, objective determination of lymphocyte subpopulations and their distribution/uniformity within tumor tissues remains largely unexplored. Here, we simultaneously measured diverse TIL subpopulations using quantitative immunofluorescence (QIF) in different areas of breast tumors to determine the heterogeneity of TILs and its possible impact for use as biomarker. Methods: Using a multiplexed QIF-based assay for simultaneous detection of DAPI (all cells), Cytokeratin (epithelial cells, M3515-DAKO), CD3 (T lymphocytes, E272--Novus), CD8 (cytotoxic T cells, C8/144B--DAKO), and CD20 (B cells, clone L26-DAKO), we measured the levels of TIL subpopulations in whole tissue section slides of 3 tumor cores obtained from different areas of 31 breast carcinomas. The levels of the markers were measured using the AQUA method of QIF and the heterogeneity was studied using numerical correlations of log2 transformed scores and variance component analysis with linear mixed effects (LME). The concordance (kappa index [κ]) between binarized scores obtained measuring 1 vs 3 cores of the same tumor was also evaluated. Results: As expected, we found a positive correlation between CD3 and CD8 levels across all patients (Pearson correlation coefficient [CC]=0.827). The levels of CD3 and CD8 showed weaker association with CD20 signal (CC=0.446 and 0.363, respectively). For all the TIL markers, the intra-tumor variation was higher than the inter-tumor differences with intraclass correlation coefficients (ICC) of 0.411 for CD3, 0.324 for CD8, and 0.252 for CD20. In the variance component analysis, 66-69% of the variance was attributable to signal differences between areas of the same tumor core and 30-33% was due to differences between cores from different areas. Consistent with this and using the median score as cutpoint to stratify cases in high/low marker levels, the concordance of measuring TILs in 1 vs 3 cores of the same tumor was κ=0.705 for CD3, κ=0.655 for CD8, and κ=0.603 for CD20. Conclusion: Objective measurement of TIL markers indicates that T and B lymphocytes show heterogeneity in breast cancer. The tumor variation of the markers is driven predominantly by differences within the same tumor core. The data from our study suggests that although a single core biopsy of tumors provides considerable information regarding the degree of lymphocyte infiltration in breast cancer patients, caution should be taken when using this as a clinical biomarker. Citation Format: Mani NL, Schalper K, Hatzis C, Chagpar A, Pusztai L, Rimm DL. Heterogeneity of tumor infiltrating lymphocytes in breast cancer and its impact for use as a biomarker. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-09.