Immune Cell Staining Research Articles

RUSSCO-RSP study of immunohistochemistry (IHC) diagnostic assays for PD-L1 expression in urothelial cancer (UC).

489 Background: This collaborative Russian Society of Clinical Oncology (RUSSCO) and Russian Society of Pathology (RSP) study assess the extent of concordance between three validated, commercially available PD-L1 IHC assays for UC patients. Methods: Tumors from 100 UC patients were stained with SP142 (Ventana), 22C3 (Dako) and SP263 (Ventana) clones as used in the clinical trials of second-line therapy of atezolizumab, pembrolizumab and durvalumab. Four trained pathologists independently evaluated the percentages of tumor (TC) and tumor infiltrating immune cells (IC) staining positive at any intensity. One test-specific cutoff rule for each assay in this analysis was pre-specified as: IC ≥5% for SP142, TC+IC ≥10% or TC ≥10% for 22C3, and TC ≥25% or IC ≥25% for SP263. Results: 300 IHC slides were scored. Patients were predominantly male (89%) with T1 (45%) and T2 (45%) stages, respectively. The percent of IC staining across 3 assays is shown to be higher than for TC staining (45% vs. 8% for SP142; 55% vs. 24% for 22C3; 72% vs. 27% for SP263). Pearson Correlation Coefficients for IC were: 0.5, 0.69 and 0.85 between SP142/22C3, SP263/22C3 and SP142/SP263, respectively. Pearson Correlation Coefficients for TC were: 0.93, 0.99 and 0.91 for the same pairs. Table indicates the agreement analysis using recommended individual cutoffs for each test. Conclusions: If a patient with UC is classified as negative by one of the three single tests using the corresponding recommended cutoff rule, the patient will be classified as negative by any other test with a high likelihood (91%-100%), and, therefore, repeated testing can be avoided.[Table: see text]

Abstract B073: PD-L1 expression in urothelial cancer: first results from RUSSCO-RSP assay comparision study

Abstract Background: To compare similarities and differences between PD-L1 IHC assays in patients with urothelial cancer (UC), collaborative Russian Society of Clinical Oncology (RUSSCO) and Russian Society of Pathology (RSP) study was conducted. The first results, presented here, describe a rate of PD-L1 IHC expression assessed by each test and evaluate concordance between 3 assays. Methods: 100 UC tumors were stained with PD-L1 IHC assays (clones SP142, 22C3 and SP263) as used in the clinical trials of second-line therapy of atezolizumab (Ventana SP142), pembrolizumab (Dako 22C3), and durvalumab (Ventana SP263). Four trained pathologists certified by Ventana/Roche and Dako/Agilent in interpreting respective assays independently evaluated the percentages of tumor (TC) and tumor-infiltrating immune cells (IC) staining positive at any intensity. Single clinical cut-off for inclusion in this comparative analysis was used: IC ≥5% for SP142, TC+IC/TC ≥10% for 22C3, and TC or IC ≥25% for SP263. Results: Patients were predominantly male (89%), 47/100 (47%) had grade 3 tumors, 45/100 (45%) and 45/100 (45%) had T1 and T2 stages, respectively. 300 IHC slides were scored. The percent of immune cell staining across 3 assays appears to be higher than for tumor cell staining (45% vs. 8% for SP142; 55% vs. 24% for 22C3; 72% vs. 27% for SP263). There were variabilities in immune cell staining for all assays and in tumor cell staining across SP142 and other assays. The percentage of PD-L1-positive patients was comparable with SP142 (9%), 22C3 (11%) and SP263 (7%). Table indicates the number of cases that were concordant with the index assay scoring algorithm, when an alternative cut-off was used to determine the allocation of cases to clinical groups above and below the cut point. Table Assay clone used for slide stainingScoring AlgorithmSP142IC ≥5%22C3TC+IC / TC ≥10%SP263TC or IC ≥25%SP142100/100 (100%)92/100 (92%)95/100 (95%)22C395/100 (95%)100/100 (100%)95/100 (95%)SP26396/100 (96%)95/100 (95%)100/100 (100%) Conclusions: First comparison showed that concordance was more than 90% between SP142, 22C3, and SP263 assays used for evaluation of PD-L1 expression in patients with urothelial carcinoma. Further analysis is required to draw a final conclusion. Citation Format: Larisa Zavalishina, Patritsiya Povilaitite, Yulia Andreeva, A. Petrov, Inna Pugach, Ekaterina Kharitonova, Alexey Rumyantsev, Grigory Raskin, Georgy Frank, Sergei Tjulandin, Ilya Tsimafeyeu. PD-L1 expression in urothelial cancer: first results from RUSSCO-RSP assay comparision study [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B073.

IDO expression in breast cancer: an assessment of 281 primary and metastatic cases with comparison to PD-L1

The immune inhibitory enzyme indoleamine 2,3-dioxygenase (IDO) has been associated with immune evasion in numerous malignancies and may mark these cancers as susceptible to anti-IDO therapies. We herein address IDO expression in breast cancers, examine the relationship between IDO and PD-L1, and investigate IDO fidelity across breast cancer primaries and metastases. IDO and PD-L1 expression was assessed in tissue microarrays containing 242 invasive primary breast cancers, 20 nodal metastases, and 19 distant metastases. IDO and PD-L1 were scored by extent in the tumor cells and immune infiltrate. Tumor IDO staining was seen in 14% of primaries including 38% of triple-negative cancers. IDO immune cell staining was seen in 14% of primaries and 29% of triple-negative cancers. Tumoral IDO and PD-L1 co-expression was seen in 8% of primaries, including 70% of tumoral PD-L1-positive cases. Immune IDO and PD-L1 co-expression was identified in 14% of primaries, including 48% of immune PD-L1-positive cases. Tumoral and immune cell IDO was conserved in 94% of matched primary/metastasis. In summary, IDO expression is common among high-grade, triple-negative breast cancers and is frequently associated with PD-L1 co-expression, suggesting that IDO might be a mechanism of anti-PD-1/PD-L1 immunotherapy resistance and that dual therapy may be of utility. Tumoral and immune cell IDO expression shows fidelity between primary and metastatic sites in treatment-naïve cancers, arguing against IDO as an independent driver for metastatic spread. Clinical trials are needed to assess the efficacy of IDO inhibition relative to IDO expression, as well as its possible role in combination with anti-PD-1/PD-L1 immunotherapy.

Comparison of Four PD-L1 Immunohistochemical Assays in Lung Cancer.

Four different programmed death ligand 1 immunohistochemical assays are approved or in development as companion or complementary diagnostics to different immunotherapeutic agents in lung carcinoma. We sought to determine whether these assays are technically equivalent and whether one antibody can be used on an alternate staining platform. Serial sections of tissue microarrays constructed from 368 cases of resected lung cancer were stained for 22C3 and 28-8 on the Dako Link 48 platform (Dako, Carpinteria, Ca) and for SP142 and SP263 on the Ventana Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ) strictly as per product insert. A protocol was developed to use the 22C3 antibody on the Ventana Benchmark Ultra platform. Differences in mean tumor cell and immune cell staining were observed between the four assays (p < 0.001). Differences between 22C3 and 28-8 were not statistically significant. Concordance of tumor cell scores was good (intraclass correlation coefficient [ICC]= 0.674), particularly when SP142 was excluded as an outlier (ICC= 0.755). The highest concordance was seen between 22C3 and 28-8 (ICC= 0.812). Concordance was poor for immune cell staining (ICC= 0.212). When dichotomized according to clinically relevant cutoffs, pairwise comparisons showed poor to moderate concordance (κ= 0.196-0.578), with positive percent agreement ranging from 15.1% to 90.0%. The 22C3 antibody performed comparably on the Dako Link 48 platform and the alternate Ventana Benchmark Ultra platform (ICC= 0.921, κ= 0.897). Concordance between the four programmed death ligand 1 immunohistochemical assays when performed and scored as intended show that apart from 28-8 and 22C3, they cannot be used interchangeably in clinical practice. A protocol was successfully developed to use 22C3 on an alternate platform, which may help to overcome some barriers to implementation.

Targeting the Programmed Cell Death-1 Pathway in Genitourinary Tumors: Current Progress and Future Perspectives.

Immune checkpoint inhibitors have revolutionized the treatment of many malignancies with over a dozen new United States Food and Drug Administration (FDA) approvals in the past six years. Due to the combination of potent treatment success and potentially deadly adverse effects from immune checkpoint inhibitors, gathering prognostic and predictive information about FDA-indicated tumors is prudent. PD-L1 expression is a poor prognostic factor and predictive of better responses from both PD-1 and PD-L1 inhibitors in a variety of tumor types including Renal Cell Carcinoma (RCC) and urothelial carcinoma. Each FDAapproved PD-1/PD-L1 drug is paired with a PD-L1 Immunohistochemistry (IHC) assay. The majority of PD-1/PDL1 inhibitor clinical trials use proprietary IHC antibodies with undefined validation data. Thus, there is need for improved knowledge and application of PD-1/PD-L1 IHC biomarkers. There is a wealth of recent publications using antibody clones to characterize tumor PD-1/PD-L1 expression profiles. PD-1 is expressed on lymphocytes. PD-L1 is expressed on both tumor cells and immune cells. IHC staining appears in membranous fashion. A cutoff of at least 5% tumor cell PD-L1 staining for positivity has worked for most studies. Caution should be observed when employing tissue microarray techniques. RCC has been the most studied of the genitourinary malignancies for PD-L1 expression. The atezolizumab- approved IHC assay is unique in that only immune cell staining is quantified for the use of this assay in urothelial carcinoma. With familiarity of the current FDA guidelines, published medical literature, and general immunohistochemical considerations, the use of immune checkpoint biomarkers can continue to flourish.

Relationship of the Breast Ductal Carcinoma In Situ Immune Microenvironment with Clinicopathological and Genetic Features.

Purpose: The immune microenvironment of breast ductal carcinoma in situ (DCIS) has yet to be fully explored, and the relationship of immune cells to genetic features of DCIS is unknown.Experimental Design: We quantified tumor associated lymphocytes (TIL) and evaluated PD-L1 protein levels by immunohistochemistry in a cohort of pure DCIS (138 and 79 cases, respectively), some of which had copy number (n = 55) and mutation data (n = 20).Results: TILs were identified in the stroma surrounding DCIS (119/138, 86%) and present at a median TIL score of 5% (range, 0%-90%). Most DCIS were negative for tumor cell PD-L1 staining (89%), but 25% of cases were positive for immune cell staining. We observed that, as in invasive breast cancer, TILs and PD-L1 positivity were significantly greater in high-grade (P = 0.002/0.035), ER-negative (P = 0.02/0.02), and ERBB2-amplified tumors (P < 0.001/0.048). Comedo necrosis was significantly positively associated with TILs (P < 0.0001) but not with PD-L1. The TILs score was significantly higher in cases with TP53 mutation (P = 0.03) but not with PIK3CA or GATA3 mutation. In the cases with copy number data, both the fraction of the genome altered and the number of telomeric imbalances were significantly positively correlated with TILs (both P < 0.001). This result strongly contrasted with invasive breast cancer data, where aneuploidy was not correlated to TIL levels.Conclusions: Although a small cohort, our data suggest a preliminary model by which the progression of DCIS to invasive carcinoma may involve an altered relationship of tumor copy number with the immune microenvironment, possibly by the immunoediting of the tumor. Clin Cancer Res; 23(17); 5210-7. ©2017 AACR.

Abstract 655: Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples

Abstract Immunohistochemistry (IHC) staining of non-small cell lung cancer (NSCLC) samples for programmed cell death ligand 1 (PD-L1) can help identify patients that may benefit from anti-PDL1 therapy. However, resection or biopsy samples cannot be obtained for some patients with NSCLC. Fine needle aspiration (FNA) is a less invasive method for obtaining samples in such patients, and it is yet to be determined if PD-L1 can be assessed using cytological samples. In this study we had three objectives 1. Develop staining protocols on the VENTANA BenchMark ULTRA automated staining platform for cytology samples fixed with the most common methods using cell lines as a model. 2, Determine the optimal cytology fixation method for VENTANA PD-L1 (SP142) IHC Assay (PD-L1 (SP142)) staining, and 3. Assess PD-L1 expression in tumor cells (TC) and tumor-infiltrating immune cells (IC) in cytological samples prepared from NSCLC patients. KARPAS 299 cell line was used as a model system for the fixation studies. Liquid-based preparations (LBPs) fixed in PreservCyt, conventional smears (unstained and pap-stained) fixed in 95% ethanol, and paraffin embedded cell blocks (CBs) fixed in 95% ethanol, 10% neutral buffered formalin (NBF), PreservCyt, and SurePath preservative were optimized on the BenchMark ULTRA using PD-L1 (SP142) antibody and OptiView DAB Detection and Amplification Kits. NSCLC FNA CBs fixed in 10% NBF (N=69) and a subset of FNAs with matched resections (N=20) were stained with the optimized protocol. FNAs and resections were assessed for percentage of TC and IC with PD-L1 staining. Staining parameters were optimized on the BenchMark ULTRA for all sample types tested, and they all produced a range of moderate to strong PD-L1 expression. CBs fixed in 95% ethanol and 10% NBF produced the highest percentage of staining, with expression in 90% of cells. CBs fixed in PreservCyt and SurePath preservative had a lower percentage of staining, 40% and 75% respectively. LBPs had 25% cells staining and smears ranged from 20%-60%. NSCLC FNA CBs fixed in 10% NBF produced interpretable results when stained with PD-L1 (SP142). IC staining was seen in 8.7% (6/69) of samples, TC staining was seen in 8.7% (6/69) of samples, both IC and TC staining was seen in 14.5% (10/69) of samples. The subset of FNAs with matched resections showed concordance with PD-L1 IC staining in 60% (12/20) of samples and TC staining in 75% (15/20) of samples. The discordant cases showed that FNAs were negative for IC when the resection was negative and positive for TC in 5% of cases when the resection was negative. The VENTANA PD-L1 (SP142) Assay staining parameters were found to be optimal for staining NSCLC FNA CB fixed in 10% NBF. PD-L1 staining was detected in both TC and IC in FNA samples and concordance of FNA and matched resections was high for both IC and TC. A larger study is necessary to validate the use of FNAs for assessment of PD-L1 expression in a clinical setting. Citation Format: Christine Boyiddle, Mark Ruboyianes, Ed Del Valle, Lukas Bubendorf, Kerstin Trunzer, Judith Pugh, Jennifer Boone. Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples [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 655. doi:10.1158/1538-7445.AM2017-655

Abstract 5623: A comparative study of four PD-L1 immunohistochemical assay in non-small cell lung cancer and evaluation of scoring system

Abstract Background: Clinical trials of immune checkpoints modulators, including both programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors, have recently shown promising activity and tolerable toxicity in pre-treated and first line NSCLC patients. At least five PD-L1 IHC assays with custom reagents and scoring-criteria are evaluated in clinical trials. However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized and the role of PD-L1 expression as a biomarker is still controversial. Materials and Methods: Retrospectively collected formalin-fixed paraffin-embedded whole-tissue sections (WTS) from 97 NSCLC cases from Seoul National University Bundang Hospital were obtained. We tested the samples divided “training set” and “validation set”: A set of 50 NSCLC WTS (32 adenocarcinoma and 18 squamous cell carcinoma) resected from 2010 to 2011 was designated “training set” and the other set of 47 NSCLC WTS (32 adenocarcinoma, 14 squamous cell carcinoma, and 1 pleomorphic carcinoma) resected from 2015 to 2016 was designated “validation set”. We examined PD-L1 protein expression using four different antibodies (Clone E1L3N, SP142, SP263 and 22C3) using Ventana XT platform with optiview detection kit for E1L3N, SP142 and SP263, and DAKO link 46 platform with pharmDx kit for 22C3 by immunohistochemical analysis. Staining of tumor cells (Tumor proportion score, TPS) and immune cells were evaluated. Results: PD-L1 protein expression was observed in 18 %, 16%, 11% and 12% of total tumors with E1L3N with Ventana optiview kit (5% cutoff), SP142 with Ventana optiview kit with amplification (1% cutoff), SP263 with Ventana optiview kit (25% cutoff) and 22C3 with DAKO PharmDx kit (50% cufoff), respectively. There was no different positive rate between “training” and “validation” set, which means there was no effect of the age of paraffin block on PD-L1 expression. Regarding staining proportion of tumor cells, 22C3 yields more stained tumor cells (highest TPS) and SP142 consistently labels fewer tumor cells (lowest TPS). Among 38 cases which were positive in one or more assays, 28.9% showed discrepant results for PD-L1 expression between the assays according to each criterion. When the same criterion applied for all assays, discrepant rate is higher (63.1 % with 1% cutoff, 71.0% with 5% cutoff). Conclusion: Although comparative determination of PD-L1 protein levels in NSCLC reveals inter-assay variability of tumor cell staining, positive rate is similar under each criterion. Therefore, there is a potential for different diagnostic results according to the key clinical cut-offs if assays and algorithms are mismatched and we should be careful when interpret the PD-L1 expression with different assays. Citation Format: Hyojin Kim, Hyun Jung Kwon, Soo Young Park, Eunhyang Park, Jin-Haeng Chung. A comparative study of four PD-L1 immunohistochemical assay in non-small cell lung cancer and evaluation of scoring system [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 5623. doi:10.1158/1538-7445.AM2017-5623

PD-L1 protein expression in tumour cells and immune cells in mismatch repair protein-deficient and -proficient colorectal cancer: the foundation study using the SP142 antibody and whole section immunohistochemistry

AimsRoutine application of PD-L1 immunohistochemistry (IHC) in colorectal cancer (CRC) is limited due to lack of standardized scoring criteria, antibody clones, and intratumoral staining heterogeneity. We assessed PD-L1 protein expression...

Concordance of tumor cell (TC) and immune cell (IC) staining with Ventana SP142, Ventana SP263, Dako 28-8 and Dako 22C3 PD-L1 IHC tests in NSCLC patient samples.

e14503 Background: PD-1/PD-L1 directed antibodies are emerging as effective oncology therapeutics with multiple diagnostic PD-L1 tests available for patient segmentation. It is essential to compare these assays to understand the risk if assays are used interchangeably. Previous data showed strong concordance (OPA &gt;85%) of TC PD-L1 expression at multiple clinically relevant cutoffs with the Ventana SP263, Dako 28-8 and 22C3 assays (Ratcliffe et al. Clin Cancer Res 2017; Ratcliffe et al. ESMO 2016 (955PD)). This work supplements previous data with concordance of IC scoring and data from Ventana SP142. Methods: IC PD-L1 IHC expression data were generated from 493 clinical NSCLC biopsy samples stained with Ventana SP263 and Dako 22C3 and 28-8 assays. In addition, consecutive sections from 200 blocks of the 493 samples were later sectioned and stained with SP142 and SP263. Concordance at matched cutoffs between the 4 assays is reported for IC, and concordance between SP263 and SP142 for TC. Results: Strong agreement in IC scoring between the SP263 and 22C3 and 28-8 assays is observed. The SP142 assay showed lower IC concordance and reduced TC PD-L1 staining/concordance vs SP263 (Table). Conclusions: Analytical similarity is shown between the SP263, 28-8 or 22C3 assays when measuring TC or IC PD-L1 expression, with high concordance between patient classification at matching TC cutoffs up to ≥50% or IC up to ≥10%, strengthening the evidence that these 3 assays could be used interchangeably at relevant clinical cutoffs. The SP142 assay shows clear analytical differences in both IC and TC staining. [Table: see text]

Assessment of heterogeneity of PD-L1 expression in NSCLC, HNSCC, and UC with Ventana SP263 assay.

e14502 Background: Clinical assessment of PD-L1 status relies on the result of testing 1 FFPE section per patient (pt). Heterogeneity of PD-L1 expression within a single tissue block and between paired blocks from the same pt could impact pt classification. This study characterizes the intra-block and intra-case heterogeneity using the VENTANA PD-L1 (SP263) Assay in non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC) for tumor cell (TC) and immune cell (IC) staining. Methods: Two blocks from the same tumor for 5 cases of each of 3 indications (30 unique blocks) representing a range of PD-L1 staining were commercially acquired and sectioned serially at 4 μm. Tumor continuity throughout the blocks was confirmed by staining sections 1 and 51 with H&amp;E. Sections 2, 25 and 50 were stained with VENTANA SP263 and scored by a single trained pathologist, using the SP263 scoring algorithm and interpretation guide. Total percentages of PD-L1+ TCs and ICs were determined. Multiple diagnostic cutoffs were applied to TC and IC scores from multiple sections within each block and pair of blocks to establish overall percentage agreement (OPA) within and between blocks at each cutoff. Additional data were available in NSCLC for 200 cases with repeat SP263 staining (minimum separation 70 μm). Results: TC PD-L1 status at all cutoffs showed no intra-block (sections ~100 μm apart) or intra-case heterogeneity (Table). IC PD-L1 status showed limited intra-block and intra-case heterogeneity, with IC % staining in discordant sections varying by no more than 5%. Results are supported by the 200 NSCLC cases, with a minimum TC OPA across cutoffs of 91% (78.5% for ICs). Conclusions: Intra-block and intra-case concordance of TC PD-L1 status is high, and IC PD-L1 agreement is good across cutoffs. This supports the reproducibility of pt PD-L1 status in NSCLC, HNSCC and UC, within and between blocks. [Table: see text]

Concordance of tumor and immune cell staining with Ventana SP142, Ventana SP263, Dako 28-8 and Dako 22C3 PD-L1 tests in NSCLC patient samples.

7 Background: PD-1/PD-L1 directed antibodies are emerging as effective therapeutics in multiple oncology settings. Multiple diagnostic PD-L1 tests are being used to understand how PD-L1 expression predicts response to these therapies. It is vital to compare these assays to understand the risk if assays are used interchangeably. Tumor cell (TC) PD-L1 expression by the Ventana SP263 and the Dako 28-8 and 22C3 assays has proven concordant at multiple clinically relevant cut offs.(Ratcliffe et al AACR 2016 [LB-094]); (Ratcliffe et al ESMO 2016 [955PD]). The current study builds on the previous study by inclusion of immune cell (IC) scoring and the Ventana SP142 assay. Methods: 493 tumor biopsy samples from NSCLC patients stained with the Ventana SP263 and Dako 22C3 and 28-8 were scored for IC PD-L1 expression, calculated as % of infiltrating IC expressing PD-L1 at any intensity. A subset of 200 tumor blocks from this cohort were sectioned and stained with SP142 and serial sections restained with SP263 to ensure an appropriate comparison. Concordance between IC and TC between these 2 assays is reported. Results: There was strong agreement in IC scoring with the Ventana SP263 and Dako 22C3 and 28-8 assays. The SP142 assay showed lower concordance for IC and reduced TC PD-L1 staining/concordance vs SP263 (Table). Conclusions: This study shows analytical similarity between the Ventana SP263, Dako 28-8 or 22C3 assays when measuring TC or IC PD-L1 expression, and high concordance between patient classification at matching TC or IC cut offs. This strengthens the evidence that these 3 assays could be used interchangeably. The Ventana SP142 assay shows clear analytical differences for both IC and TC staining, suggesting it is not appropriate to interchange with the other assays. [Table: see text]

Immune cell phenotyping of clear cell renal cell carcinoma.

511 Background: Clear cell renal cell carcinoma (ccRCC) tumors develop mechanisms that impair function and/or prevent entry of the host infiltrating immune cells (immune exclusion) within the tumor microenvironment. The goal of immunotherapy is to overcome this immune resistance. We aim to characterize the T−cell populations in a cohort of largely untreated high risk patients with ccRCC. Methods: We prospectively collected ccRCC tumor and adjacent normal kidney (NK) from patients undergoing surgical resection at our institution from 6/2015-8/2016. Immune cell phenotyping was performed by immune cell staining of single cell suspensions. Analysis of immune cell populations were determined by CD45+ staining and corresponding proportions of different T−cell populations (CD3+, CD4+, CD4+Treg, and CD8+ T cells). Staining for CD4+Treg was not available for two patients. Student t−test was utilized to compare the immune populations between tumor and adjacent NK tissue. Analysis was also conducted by stratifying patients who presented with localized versus metastatic disease. Results: A total of 31 tumor and adjacent NK were analyzed. Median tumor pathological size was 8.5cm (2.9cm−18cm), 27(87%) had pT3a−pT3b and 13(42%) presented with metastatic disease. Overall 84% of tumors had higher immune infiltrate with an average ratio of four-fold increase compared to adjacent NK as determined by CD45+ cells. Intriguingly, the other 16% presented with metastatic (4) or rapidly metastatic disease (1). Orthogonal validation with inferred immune populations using RNAseq data from the The Cancer Genome Atlas (TCGA) demonstrated similar aggressive behavior in tumors with lower immune infiltrate compared to NK. Comparison of immune cell populations of tumor and NK tissue is shown in table 1. No specific T-cell subtype was associated with specific clinical outcomes in this cohort. Conclusions: Our data shows a general trend of immune infiltration in ccRCC when compared to adjacent NK with a diversity of T-cell subsets and possible evidence of immune exclusion. Further genomic characterization of these tumors is currently underway. [Table: see text]

PD-L1 Immunohistochemistry Assays for Lung Cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project

IntroductionThe Blueprint Programmed Death Ligand 1 (PD-L1) Immunohistochemistry (IHC) Assay Comparison Project is an industrial-academic collaborative partnership to provide information on the analytical and clinical comparability of four PD-L1 IHC assays used in clinical trials. MethodsA total of 39 NSCLC tumors were stained with four PD-L1 IHC assays (22C3, 28-8, SP142, and SP263), as used in the clinical trials. Three experts in interpreting their respective assays independently evaluated the percentages of tumor and immune cells staining positive at any intensity. Clinical diagnostic performance was assessed through comparisons of patient classification above and below a selected expression cutoff and by agreement using various combinations of assays and cutoffs. ResultsAnalytical comparison demonstrated that the percentage of PD-L1–stained tumor cells was comparable when the 22C3, 28-8, and SP263 assays were used, whereas the SP142 assay exhibited fewer stained tumor cells overall. The variability of immune cell staining across the four assays appears to be higher than for tumor cell staining. Of the 38 cases, 19 (50.0%) were classified above and five (13%) were classified below the selected cutoffs of all assays. For 14 of the 38 cases (37%), a different PD-L1 classification would be made depending on which assay/scoring system was used. ConclusionsThe Blueprint PD-L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD-L1 expression for three assays, interchanging assays and cutoffs would lead to “misclassification” of PD-L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy-related PD-L1 cutoffs.

Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142. E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

IntroductionA substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC).ResultsThe expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium.ConclusionTaken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

Neonatal exposure to mild hyperoxia causes persistent increases in oxidative stress and immune cells in the lungs of mice without altering lung structure

Preterm infants often require supplemental oxygen due to lung immaturity, but hyperoxia can contribute to an increased risk of respiratory illness later in life. Our aim was to compare the effects of mild and moderate levels of neonatal hyperoxia on markers of pulmonary oxidative stress and inflammation and on lung architecture; both immediate and persistent effects were assessed. Neonatal mice (C57BL6/J) were raised in either room air (21% O2), mild (40% O2), or moderate (65% O2) hyperoxia from birth until postnatal day 7 (P7d). The mice were killed at either P7d (immediate effects) or lived in air until adulthood (P56d, persistent effects). We enumerated macrophages in lung tissue at P7d and immune cells in bronchoalveolar lavage fluid (BALF) at P56d. At P7d and P56d, we assessed pulmonary oxidative stress [heme oxygenase-1 (HO-1) and nitrotyrosine staining] and lung architecture. The data were interrogated for sex differences. At P7d, HO-1 gene expression was greater in the 65% O2 group than in the 21% O2 group. At P56d, the area of nitrotyrosine staining and number of immune cells were greater in the 40% O2 and 65% O2 groups relative to the 21% O2 group. Exposure to 65% O2, but not 40% O2, led to larger alveoli and lower tissue fraction in the short term and to persistently fewer bronchiolar-alveolar attachments. Exposure to 40% O2 or 65% O2 causes persistent increases in pulmonary oxidative stress and immune cells, suggesting chronic inflammation within the adult lung. Unlike 65% O2, 40% O2 does not affect lung architecture.

PD-L1 Expression as a Predictive Biomarker in Cancer Immunotherapy.

The resurgence of cancer immunotherapy stems from an improved understanding of the tumor microenvironment. The PD-1/PD-L1 axis is of particular interest, in light of promising data demonstrating a restoration of host immunity against tumors, with the prospect of durable remissions. Indeed, remarkable clinical responses have been seen in several different malignancies including, but not limited to, melanoma, lung, kidney, and bladder cancers. Even so, determining which patients derive benefit from PD-1/PD-L1-directed immunotherapy remains an important clinical question, particularly in light of the autoimmune toxicity of these agents. The use of PD-L1 (B7-H1) immunohistochemistry (IHC) as a predictive biomarker is confounded by multiple unresolved issues: variable detection antibodies, differing IHC cutoffs, tissue preparation, processing variability, primary versus metastatic biopsies, oncogenic versus induced PD-L1 expression, and staining of tumor versus immune cells. Emerging data suggest that patients whose tumors overexpress PD-L1 by IHC have improved clinical outcomes with anti-PD-1-directed therapy, but the presence of robust responses in some patients with low levels of expression of these markers complicates the issue of PD-L1 as an exclusionary predictive biomarker. An improved understanding of the host immune system and tumor microenvironment will better elucidate which patients derive benefit from these promising agents.

Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×107 PFU) or CPXV (>102 PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.

Reversal of T Cell Exhaustion in Pre-Treatment Marrow T Cells Is Associated with Effective Graft-Versus-Leukemia Responses to Donor Lymphocyte Infusion

Abstract 1903Donor lymphocyte infusion (DLI) can provide curative treatment for relapsed hematologic malignancies following allogeneic hematopoietic stem cell transplant (HSCT). However, the precise mechanism by which DLI eliminates leukemia cells in vivo has not been established. We hypothesized that marrow-infiltrating immune populations play a critical role in DLI responses since marrow is the primary site of disease and a reservoir of high-avidity antigen-specific memory T cells that can recognize tumor antigens, therefore potentially mediating graft-versus-leukemia (GvL) responses.We performed immunohistochemical staining of immune cells in serial marrow biopsies collected before and after DLI from 29 patients with relapsed CML. Twenty-two patients achieved cytogenetic remission within twelve months (‘responders') while 7 patients demonstrated persistent disease (‘non-responders'). While no significant changes in the numbers of circulating T cells were seen between patient groups following DLI, the median number of marrow-infiltrating CD8+ T cells increased 2-fold in responders but remained unchanged in non-responders (P=0.02), demonstrating that clinical response to DLI is associated with T cell responses at the site of disease that may not be apparent in the peripheral blood.To investigate whether immune cell infiltration of the marrow could predict DLI response, we compared pre-treatment samples from both patient groups. Responders exhibited significantly higher proportions of CD8+ T cells in pre-DLI marrow compared to non-responders (5% vs 2.5%; P=0.01). Because disease burden is a known risk factor for ineffectual DLI response, we evaluated the interaction between disease burden and pre-existing CD8+ T cell infiltrate through the clinical course of 8 patients with high (≥70%) pre-treatment marrow cellularity. Three of 8 had ≥5% CD8+ T cell marrow infiltrates, and all 3 subsequently achieved cytogenetic remission. In contrast, 5 of 8 patients had <5% CD8+ T cell infiltrates, and all failed to show a cytogenetic response. ROC analysis of the entire original patient cohort using disease burden and CD8+ T cell infiltrate criteria (pre-DLI cellularity ≤70% or CD8+ T cell ≥5%) revealed a sensitivity and specificity of 100% in predicting responsiveness to DLI. These findings highlight the use of CD8+ T cell marrow infiltration as a new predictive marker for DLI response and suggest that immune status can overcome the adverse influence of high disease burden in therapeutic response.To identify candidate mechanisms underlying T cell responses to DLI, we performed mRNA expression profiling of CD3+ T cells isolated from matched pre- and post-treatment marrows of 4 responders and 2 non-responders (U133+ Affymetrix cDNA microarrays). We first compared global gene expression patterns between pre-treatment marrow-infiltrating T cells of both groups. Notably, 28% of significantly upregulated genes in responders play critical roles in T cell exhaustion. This finding was strengthened by unbiased gene set enrichment analysis (GSEA) using 880 sets from the Molecular Signatures Database spiked with 17 additional specifically curated T cell exhaustion sets. Four of 15 positively enriched sets were T cell exhaustion-specific. We next compared differential gene expression in marrow-infiltrating T cells before and after therapy in responders compared to non-responders and found 21% of significantly down-regulated genes to be components within T cell exhaustion pathways along with repression of multiple, distinct T cell exhaustion gene sets. The robust reversibility of T cell exhaustion signatures after DLI therapy underscores this gene module as a likely therapeutic target of DLI.In conclusion, CD8+ T cell marrow infiltration may serve as a novel predictive marker of response to DLI, including patients with high disease burden. In addition, these data implicate T cell exhaustion in distinguishing responders from non-responders and, provocatively, propose reversal of T cell exhaustion as a potential marker of DLI responsiveness in patients with relapsed CML after HSCT. These studies illustrate the importance of local immune responses at the site of disease and suggest a potential tool to predict DLI response in other hematologic malignancies. The clinical debut of newer agents that reverse T cell exhaustion suggests their use in lieu of DLI to promote GvL responses after allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.