PD-L1 Immunohistochemistry Research Articles

P2.09-15 PD-L1 Expression and Lymphocyte Infiltration in Resected Stage IIIAN2 NSCLC: Preliminary Data from a Lung ART Ancillary Study

Patients with resectable stage IIIA N2 NSCLC, are at high risk of both systemic and loco-regional relapse following surgical resection, necessitating neo-adjuvant or adjuvant treatments. Prognostic biological markers are needed. Parameters from the immune microenvironment, including PD-L1 expression and lymphocytic infiltration, have been poorly described in this group of patients. Thus we assessed simultaneously PD-L1 expression and TIL density in a cohort of stage IIIA N2 Lung ART patients, and correlated the results with clinical and pathological features before adjuvant treatment. Formalin fixed paraffin-embedded tumor surgical specimens from 247 patients included in the Lung Adjuvant Radiotherapy Trial (NCT00410683) were studied. PD-L1 immunohistochemistry was performed centrally on whole slides using a validated clinical PD-L1 assay. Expression of PD-L1 in tumor cells (TC) and immune cell (IC) was scored by a trained pathologist. Morphological assessment of TIL density (percentage of tumor area) was performed on whole hematoxylin-eosin stained slides. Surgical and pathology reports were reviewed by an independent expert committee for tumor staging. Association between immune parameters and baseline clinical characteristics were assessed in exploratory analyses in order to provide insights on immune activity in resected NSCLC patients. PD-L1 expression in ≥1% TC, ≥50% TC, ≥1% IC, ≥10% IC was observed in 47.8%, 21.9%, 61.5%, 7.3% of patients, respectively. In univariate analysis, high PD-L1 expression in both tumor cells and immune cells for all cut points correlated strongly with a higher TIL density (p-values ≤0.001). In 41 (16.6%) patients with preoperative chemotherapy (CT), a higher TIL density was observed (mean 28.1 vs. 17.5%, p=0.0018) as compared to patients without preoperative CT, but no difference was noted for PD-L1 expression in both TC and IC,. Skip N2 metastases were associated with a higher TIL infiltration (mean 22.9% vs. 17.4% p=0.014). We found no significant correlation between PD-L1 or TIL infiltration with the number of mediastinal lymph nodes stations involved on pathological examination and with histological tumor subtypes (squamous cell carcinoma vs. adenocarcinoma). PD-L1 expression levels in TC and IC appeared similar in stage IIIA N2 NSCLC as compared to other stages. Expression in both TC and IC strongly correlated with TIL infiltration, suggesting a prominently immune-induced expression mechanism. Preoperative chemotherapy was associated with a higher TIL infiltration but not higher PD-L1 expression. Patients with skip N2 metastases harbored a higher level of TIL density, a finding consistent with a more active immune microenvironment in this group of patients with better prognosis. These data will be subsequently updated on a larger number of patient and correlated to clinical follow-up.

1491P - PD-L1 expression in primary tumour vs metastatic samples in the phase III MYSTIC study in first-line metastatic (m) NSCLC

Abstract Background PD-L1 expression determined by immunohistochemistry (IHC) can be a useful biomarker to assess the likelihood of benefit with anti-PD-1/PD-L1 therapies in pts with mNSCLC. Understanding the impact of sample type (primary tumour or metastatic) on predictivity of benefit will inform the suitability of such samples for clinical testing. MYSTIC (NCT02453282) was an open-label, phase III study of durvalumab (D) ± tremelimumab vs chemotherapy (CT) as first-line treatment for mNSCLC; stratification factors for randomisation included tumour cell (TC) PD-L1 expression (≥25% vs Methods All pts enrolled in MYSTIC were assessed centrally for TC staining for PD-L1 from tissue samples acquired Results Of 1118 pts randomised, 716 (64.0%) provided a primary tumour sample and 402 (36.0%) provided a metastatic sample. The prevalence of PD-L1 TC ≥25% assessed using primary vs metastatic samples was 43.0% vs 44.8% (p = 0.569). Outcomes in pts with TC ≥ 25% determined using samples of each type are shown. Table . 1491P Primary tumour sample PD-L1 TC ≥25% Metastatic sample PD-L1 TC ≥25% Durvalumab (n = 104) Chemotherapy (n = 100) Durvalumab (n = 59) Chemotherapy (n = 62) Median OS, months 15.8 13.0 20.5 12.9 HR for OS vs CT (95% CI) 0.81 (0.59–1.11) - 0.65 (0.41–1.01) - 24-month OS, % 33.4 19.1 46.9 28.6 ORR, % 30.8 37.0 44.1 38.7 Median DoR, months Not reached 4.4 Not reached 4.1 Conclusions In MYSTIC, PD-L1 TC ≥25% prevalence was similar using primary or metastatic samples. Favourable HRs for OS with D vs CT were seen in pts with TC ≥ 25% expression as determined in either the primary tumour or a metastatic site. Results should be interpreted with caution given the retrospective nature of the analysis. Clinical trial identification NCT02453282 (release date: 25 May 2015). Editorial acknowledgement Samantha Holmes, PhD, of Cirrus Communications (Macclesfield, UK), an Ashfield company, funded by AstraZeneca and in accordance with Good Publication Practice (GPP3) guidelines. Legal entity responsible for the study AstraZeneca PLC. Funding AstraZeneca. Disclosure N. Reinmuth: Honoraria (self), Travel / Accommodation / Expenses: BMS; Honoraria (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Travel / Accommodation / Expenses: Bohringer Ingelheim; Honoraria (self), Travel / Accommodation / Expenses: Roche; Honoraria (self), Travel / Accommodation / Expenses: MSD; Honoraria (self), Travel / Accommodation / Expenses: Takeda; Honoraria (self): Pfizer. A. Boothman: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. B.C. Cho: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): AstraZeneca; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Roche; Advisory / Consultancy: BMS; Advisory / Consultancy, Research grant / Funding (self): Ono; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Yuhan; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Eli Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Janssen; Speaker Bureau / Expert testimony: Takeda; Advisory / Consultancy: MSD; Shareholder / Stockholder / Stock options: TheraCanVac Inc; Honoraria (self), Research grant / Funding (self): Bayer; Honoraria (self), Research grant / Funding (self): MOGAM Institute; Honoraria (self), Research grant / Funding (self): Dong-A ST; Honoraria (self), Research grant / Funding (self), Licensing / Royalties: Champions Oncology; Honoraria (self), Research grant / Funding (self): Dizal Pharma; Honoraria (self), Research grant / Funding (self): MSD. K.H. Lee: Research grant / Funding (institution): AstraZeneca. M. Ahn: Advisory / Consultancy, Research grant / Funding (institution), Spouse / Financial dependant: AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): AbbVie; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Research grant / Funding (institution): BIND Biosciences; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Eli Lilly; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): MedImmune; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Pfizer. M. Scott: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. J. Whiteley: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. J. Walker: Full / Part-time employment: AstraZeneca. V. Karwe: Full / Part-time employment: AstraZeneca. P. Mukhopadhyay: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. P. Thiyagarajah: Full / Part-time employment: AstraZeneca. U. Scheuring: Full / Part-time employment: AstraZeneca. N. Rizvi: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Merck; Advisory / Consultancy: Roche; Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Lilly; Advisory / Consultancy: AbbVie; Advisory / Consultancy: Merck KGaA; Advisory / Consultancy: Regeneron; Shareholder / Stockholder / Stock options: Gritstone Oncology; Shareholder / Stockholder / Stock options: ARMO Biosciences. All other authors have declared no conflicts of interest.

LBA89 - PD-L1 expression, patterns of progression and patient-reported outcomes (PROs) with durvalumab plus platinum-etoposide in ES-SCLC: Results from CASPIAN

LBA89 - PD-L1 expression, patterns of progression and patient-reported outcomes (PROs) with durvalumab plus platinum-etoposide in ES-SCLC: Results from CASPIAN

2025P - Expression of PD-L1 in Chinese patients with common cancers

BackgroundPD-L1 immunohistochemistry (IHC) data in China was less reported. We systematically investigated PD-L1 IHC data of Chinese patients generated by Origimed. MethodsPD-L1 was stained using IHC on cancer samples from 2060 Chinese patients in Origimed since 2017. Written informed consent was obtained from each patient. IHC staining was performed on FFPE tissue sections using anti-PD-L1 antibody 28-8. Tumor Proportion Score (TPS) was applied on all the samples. We investigated PD-L1 TPS in lung adenocarcinoma (n=893), lung squamous carcinoma (n=172), liver cancer (n=404), esophageal cancer (n=186), colorectal cancer (n=166), pancreatic cancer (n=136) and gastric cancer (n=103). We then further applied PD-L1 combined positive score (CPS) on the 103 gastric cancer samples. All the slides were reviewed by the same senior pathologist. 95% confidence interval (CI) was obtained by bootstrap. Information entropy (Shannon’s formula) was measured in natural units. ResultsThe highest proportion of PD-L1 TPS>= 1% was observed in lung squamous carcinoma (49%; 95% CI: [43%, 59%]), followed by lung adenocarcinoma (25%; 95% CI: [22%, 28%]), liver cancer (14%; 95% CI: [11%, 18%]), esophageal cancer (12%; 95% CI: [8.1%, 17%]), pancreatic cancer (11%; 95% CI: [5.9%, 16%]), gastric cancer (7%; 95% CI: [1.9%, 12%]) and colorectal cancer (4%; 95% CI: [1.2%, 6.6%]). The proportion of PD-L1 CPS>= 1 for gastric cancer was 17% (95% CI: [9.7%, 24%]). In gastric cancer, information entropy of TPS and CPS was 0.40 and 0.78, respectively. ConclusionsThe proportion of PD-L1 TPS>= 1% in Chinese lung squamous cell carcinoma (49%) was much higher than that in lung adenocarcinoma (25%). PD-L1 CPS contained more information than TPS in gastric cancer. Thus, we recommend applying CPS to gastric cancer in China. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureX. Pan: Full / Part-time employment: Origimed. Y. Dai: Full / Part-time employment: Origimed. D. Chen: Full / Part-time employment: Origimed. K. Wang: Full / Part-time employment: Origimed. X. Dong: Full / Part-time employment: Origimed. All other authors have declared no conflicts of interest.

P2.09-16 Assessment of PD-L1 and CD8 Expression in Lung Cancer Using RNA in Situ Hybridisation

PD-L1 is routinely assessed using immunohistochemistry (IHC) as a companion-diagnostic test. However, patients may be missing out on therapy as issues exist in determining PD-L1 positivity. This is partly due to the use of different antibodies, staining platforms and positivity cut-offs. The use of RNA in-situ hybridisation (RISH) for the detection of PD-L1 levels may be a way to address this; particularly as PD-L1 mRNA expression levels are associated with better overall survival in NSCLC. It is well established that PD-L1 expression is associated with increased numbers of Tumour Infiltrating Lymphocytes (TILs) including those which are CD8+. Therefore, it may also be worthwhile to combine PD-L1 testing with additional marker testing such as CD8. The aim of this study is to investigate the expression of PD-L1 and CD8 by RISH in a cohort of NSCLC samples and correlate with clinical outcome. RISH protocols (RNAScope® 2.5 HD assay, Advanced Cell Diagnostics, Inc.) were initially optimised on a well annotated cell line TMA (low, moderate and high expression) and compared with standard IHC (DAKO - clone 22C3). Following successful optimisation of the technique, a test cohort of 35 full-face FFPE NSCLC samples were examined for PD-L1 expression by both RISH and IHC. All slides were scored independently by a pathologist. RISH slides were scored as per ACD guidelines (0-4; based on number of positive dots per cell and number of dot clusters). IHC was scored using the standard tumour proportion score system. Image analysis (IA) was subsequently performed by Visiopharm®. Currently, a further cohort of 200 FFPE samples is being assessed, in addition to 50 FFPE samples from patients who received an anti-PD-1 therapy. Optimisation is on-going for dual CD8/PD-L1 RISH staining. In the initial 35 samples, PD-L1 was detected in 20% of cases by IHC (≥50% positivity) with mRNA expression identified in 14% of cases by RISH (≥2). In contrast with other recently published studies, issues were identified between IHC and RISH. There were 3 cases which scored positive by IHC, which were negative by RISH. Additionally, 1 case was positive by RISH but negative by IHC. The comparison between both assays produced a ‘moderate agreement’ as assessed using Cohen’s kappa coefficient (κ=0.528, p=0.002). IA undertaken on both assays could not be statistically compared using Cohen’s kappa coefficient due to the use of a composite scoring matrix to quantify RISH mRNA signals. However, IHC stained sections scored by IA showed similarity to IHC scored by a pathologist. Further optimisation is required on the RISH IA scoring algorithm. A comparison of PD-L1 IHC with RISH, combined with dual CD8/PD-L1 staining in a larger cohort of clinical samples is on-going, which will further determine the clinical utility of this technique. Data from our test cohort of samples have shown that PD-L1 detection by RISH is feasible and compares well to IHC. Preliminary data would also suggest that IA may be better than a pathologist alone, particularly in borderline cases. RISH may be a suitable method for improved detection of PD-L1 in NSCLC.

IBS15.02 Determinants of Response to ICI

While great promise has emerged with the development of immunotherapy (IT) in lung cancer, we are still struggling with how to select the right treatment to the right patients and how to select the patients, who will benefit from IT. Only 20-30 % of the patients with NSCLC will have a clear benefit from IT compared to chemotherapy alone, and the challenge is to select those patients. PD-L1 protein expression demonstrated by immunohistochemistry (IHC) has been pursuit as primary selection biomarker in the clinical trial development. However, the major challenge for evaluation of PD-L1 and top compare the results from one study to another was the use of different antibodies/assays and processing by the different companies. A comparison of the different PD-L1 assays was performed in the “PD-L1 Blueprint Project”, and three antibodies/assays was found to be performing similarly and could be interchangeable (1,2). While higher PD-L1 expression seems to be associated with gradually increased outcome of IT, a tumor proportion score (TPS) ≥ 50% seems to be useful in the choice of IT (e.g. pembrolizumab) monotherapy versus IT plus chemotherapy (CT). The results (PFS/OS) of IT monotherapy for patients with high PD-L1 expression is not significantly different than for IT+CT, although not directly compared, while for patients with tumors having lower PD-L1 expression, the combination of IT+CT seems better (3, 4). Thus, the predictive role of PD-L1 expression might be different between IT alone and IT+CT. The prognostic role of PD-L1 (e. g. association to outcome without any therapy) is still not clear with conflicting reported outcomes in the literature. Tumor mutation burden (TMB) has in some clinical trials demonstrated to be of predictive value, both based on tissue and plasma (5, 6, 7). In several studies it has been shown that the patient population with high TMB has little overlap with the patient population with high PD-L1 expression (6). Prospective clinical trials arer today performed evaluating plasma TMB as predictive biomarker (b-FIRST). However, also challenges related to TMB reports occur; different assay platforms have been used in different clinical trials, different definitions (e. g cut-offs) have been applied in the definition of high TMB vs low TMB, and also for TMB an comparison of results /assays seems to be needed. Such comparison studies are on-going both in the US (i.e. Friends of Cancer) and in Europe. On this stage, it is not clear whether TMB can replace PD-L1 IHC in treatment decisions; who will benefit from IT or not? Some studies (i.e. CheckMate 227) (8), indicate also that the two predictive assays are not “competing” but complementary. It has been shown that for patients having tumors with low (≤1%)- or no PD-L1 expression, but high TMB a treatment option of combined therapy with nivolumab+ ipilumumab seems justified. There are also conflicting results reported on the prognostic role of TMB associated to no systemic therapy or predictive role associated to chemotherapy alone. Other candidate biomarkers are under investigations and will be discussed. REFERENCES: Hirsch FR et al. J Thorac Oncol 2017 (2): 208-222 Tsao MS et al: J Thorac Oncol 2018 (9): 1302-1311 Gandhi L et al; N Engl J Med 2018 378(22): 2078-2092 Reck M et al; N Engl J Med 2016 375(19): 1823-1833 Carbone DP et a;; N Engl J Med 2017 376(25): 2415-2426 Gandara DR et al. Nat Med 2018 (9): 1441-1448 Samstein RM et al; Nat Genet 2019 (2): 202-206 Hellman MD et al; N Engl J Med 2018 378(22): 2093-2114

V-domain Ig-containing suppressor of T-cell activation (VISTA), a potentially targetable immune checkpoint molecule, is highly expressed in epithelioid malignant pleural mesothelioma.

V-domain Ig-containing suppressor of T-cell activation (VISTA) is an immune checkpoint gene that inhibits anti-tumor immune responses. Since most malignant pleural mesotheliomas do not respond to anti-programmed cell death(-ligand)1 (PD-(L)1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) therapy and given the recent finding of The Cancer Genome Atlas Study that pleural mesothelioma displays the highest expression of VISTA among all cancers studied, we examined VISTA expression in a large pleural mesothelioma cohort. VISTA and PD-L1 immunohistochemistry were performed on tissue microarray of immunotherapy-naive pleural mesotheliomas (254 epithelioid, 24 biphasic and 41 sarcomatoid) and ten whole-tissue sections of benign pleura (VISTA only). Percentages of tumor and inflammatory cells with positive staining were assessed. Optimal prognostic cutoff percentages were determined using maximally selected rank statistics. Overall survival was evaluated using Kaplan-Meier methods and Cox proportional hazard analysis. All benign mesothelium expressed VISTA. Eighty-five percent of 319 and 38% of 304 mesotheliomas expressed VISTA and PD-L1 (88% and 33% of epithelioid, 90% and 43% of biphasic, and 42% and 75% of sarcomatoid), respectively. Median VISTA score was significantly higher in epithelioid (50%) (vs. biphasic [20%] and sarcomatoid [0]) (p < 0.001), while median PD-L1 score was significantly higher in sarcomatoid tumors (20%) (vs. biphasic and epithelioid [both 0%]) (p < 0.001). VISTA and PD-L1 were expressed in inflammatory cells in 94% (n = 317) and 24% (n = 303) of mesothelioma, respectively. Optimal prognostic cutoffs for VISTA and PD-L1 were 40% and 30%, respectively. On multivariable analysis, VISTA and PD-L1 expression in mesothelioma were associated with better and worse overall survival (p = 0.001 and p = 0.002), respectively, independent of histology. In a large cohort of mesothelioma, we report frequent expression of VISTA and infrequent expression of PD-L1 with favorable and unfavorable survival correlations, respectively. These findings may explain poor responses to anti-PD-(L)1 immunotherapy and suggest VISTA as a potential novel target in pleural mesothelioma.

Immune checkpoint inhibitor associated myocarditis occurs in both high-grade and low-grade forms.

Immune checkpoint inhibitor therapy for malignancy has been associated with adverse events including myocarditis. It has been unclear if there are distinct pathologic grades of this myocarditis that are associated with distinct clinical outcomes. Cardiac tissue from ten patients with immune checkpoint inhibitor myocarditis (nine biopsies and one autopsy) were evaluated using immunohistochemistry for CD3, CD8, CD68, tryptase, PD-L1, and C4D. The immune checkpoint inhibitor myocarditis cases were classified as either high grade (>50 CD3+ cells/hpf) or low grade (≤50 CD3+ cells/hpf). The densities of macrophages, T cells, eosinophils, necrotic myocytes, and PD-L1+ macrophages and myocytes were compared between the two groups and with 13 cases of grade 2R acute cellular allograft rejection. Three patients were classified as high-grade myocarditis and seven as low grade. There were higher densities of CD3+ cells and CD8+ cells in high-grade immune checkpoint inhibitor myocarditis and rejection compared with low-grade myocarditis. The number of CD68+ macrophages was higher in high-grade myocarditis compared with low-grade myocarditis and rejection. For both grades of myocarditis, there was a higher CD68/CD3 ratio and a higher density of PD-L1+ macrophages and myocytes compared with rejection. Clinically, there were trends toward higher serum troponin levels and shorter interval from first immune checkpoint inhibitor treatment in the high-grade myocarditis group compared with the low-grade group. All the patients with high-grade myocarditis died, while all the patients with low-grade myocarditis were still living. These data suggest that immune checkpoint inhibitor myocarditis occurs in two forms, a high-grade form with increased inflammatory cell infiltration and a more fulminant clinical course, and a low-grade form with a lower degree of inflammatory cell infiltration and a more indolent clinical course. Compared with acute cellular rejection, immune checkpoint inhibitor myocarditis is characterized by a more lymphohistiocytic inflammatory infiltrate with an increased CD68/CD3 ratio and increased PD-L1+ macrophages and myocytes.

PD-L1 Immunohistochemistry on Cell Block Preparations From Different Fixatives: A Pilot Study

Abstract Objectives Immunohistochemistry especially for biomarkers such as PD-L1 in personalized medicine is increasingly being performed on cell blocks from cytopathology samples. We evaluated the effects of staining cell blocks (CBs) from different cytologic fixatives using four different commercially available PD-L1 antibody clones. Methods PD-L1 immunohistochemistry (IHC) using four different commercially available clones was performed on eight cell blocks processed from two different fixatives and also on eight formalin-fixed tissues. The clones used were 22C3 at 1:50 dilution and 1:100 dilution, SP263, SP142, and E1L3N. The cell block (CB) samples contained cells that strongly express PD-L1 and either fixed in CytoLyt (methanol-based fixative, n = 4) or CytoRich (ethanol-formalin-based fixative, n = 4). Formalin-fixed control tissues (tonsillar tissue, n = 4; stomach, n = 4) were also included. Results Expression for PD-L1 was noted on all the CytoRich fixed cell blocks (n = 4) for all four antibody clones, with the intensity and distribution of expression comparable to the formalin fixed tissues (n = 8). However, consistent absence of expression for PD-L1 was noted on all the CytoLyt fixed cell blocks (n = 4) for all four antibody clones. Conclusion The results of this pilot study demonstrate that PD-L1 IHC on cell blocks is feasible. However, there is a need to validate IHC protocols according to specific fixation methods.

PD-L1 Immunohistochemistry-Discrepant Results Between Synchronous Tumor Samples May Cause More Treatment Choice Dilemmas Than Molecular Heterogeneity in Patients With Advanced Non-Small Cell Lung Cancers.

Biomarker analyses have become mandatory for treatment choices in patients with advanced non-small cell lung cancers. PD-L1 expression for immunotherapy as well as oncogenic molecular alterations for targeted therapies must be analyzed in tumor samples. Intersample heterogeneity may cause dilemmas in treatment choices when faced with discrepant biomarker results. We led a retrospective study evaluating the potential impact on guideline-based therapeutic decisions of biomarker analyses performed in several synchronous tumor samples per patient. We collected retrospective data about patients with advanced non-small cell lung cancers, and synchronous tumor paired samples were analyzed for PD-L1 immunohistochemistry (IHC) and oncogene molecular statuses. Among 34 patients, none had a discrepant result between paired samples with respect to oncogene molecular statuses. At the opposite, intersample-discrepant PD-L1 IHC results may have caused treatment choice dilemmas for 6 (17.6%) patients discussing first-line therapy options (ie, immune checkpoint inhibitor therapy versus other systemic therapy with regard to the threshold of ≥50% PD-L1 IHC-positive tumor cells). In addition, for 6 (17.6%) other patients, different PD-L1 IHC results may have also influenced the choice of one therapy or another in second-line therapy (ie, with regard to the threshold of ≥1% PD-L1 IHC-positive tumor cells). In our case series, discrepant results with regard to PD-L1 IHC between paired tumor samples could generate more treatment choice dilemmas than the molecular heterogeneity of oncogenic drivers.

Clinical interest of PD-L1 immuno-histochemistry expression as a predictive factor of Bacillus Calmette Guerin (BCG) efficacy in refractory high-risk non-muscle-invasive bladder cancer (NMIBC).

To assess PD-L1 expression in tumor (TC) and tumor infiltrating immune cells (IC) as a predictive factor of BCG therapy failure in high-risk NMIBC. Patients treated with complete resection followed by bladder BCG instillation for high-risk NMIBC were included. Early recurrence (ER) was defined as tumor recurrence after BCG induction course. The association between ER and immuno-histochemistry PD-L1 (E1L3N clone) expression by tumors cells (TC) and tumor infiltrating immune cells (IC) was investigated using an exact Fisher test variant. A total of 186 patients were included, of whom 38 (20.4%) were ER, 35 (18.8%) were positive for TC PD-L1 expression and 60 (32.3%) were positive for IC PD-L1. ER was not significantly (p = 0.97) more frequent in the TC PD-L1 ≥ 1% group (n = 7, 20.0%) than in the TC PD-L1-negative group (n = 31, 20.5%). Patients with IC PD-L1 negative had ER in 15 (19.2%) cases and patients with IC PD-L1 ≥ 1% had ER in 23 (21.3%) cases. PD-L1-positive expression for IC (threshold > 1%) was correlated with immune infiltrate density (95.2% dense immune infiltrate vs 47.2% low immune infiltrate, p < 0.05), with increased expression of PD-L1 by IC after BCG therapy (p = 0.006). No association was observed between immuno-histochemistry PD-L1 positivity and ER after BCG therapy. Nevertheless, the relationship between immune infiltrate and PD-L1 positivity confirmed the interest of assessing the immune infiltrate density to define tumor's profile.

The expression of PD-L1 in salivary gland carcinomas

Objective was to analyze the role of PD-L1 and its relation to demographic, patho-clinical and outcome parameters in salivary gland carcinoma (SGC) patients. Patients treated for salivary gland carcinomas between 1994 and 2010 were included. A retrospective chart review for baseline characteristics, pathohistological, clinical and outcome data was performed. Immunohistochemistry for PD-L1 was performed using tissue microarrays. PD-L1 expression was assessed in tumor cells and tumor-infiltrating immune cells (TIIC) and statistical analysis with regard to baseline and outcome data was performed. Expression of PD-L1 (by means ≥1% of the cells with PD-L1 positivity) was present in the salivary gland carcinoma cells of 17%, in the TIIC of 20% and in both tumor cells and TIIC of 10% the patients. PD-L1 expression in tumor cells and both tumor cells and TIIC was related to tumor grading (p = 0.035 and p = 0.031, respectively). A trend towards higher grading was also seen for PD-L1 expression in TIICs (p = 0.058). Patients with salivary duct carcinomas and PD-L1 expressing TIICs showed a significantly worse DFS and OS (p = 0.022 and p = 0.003, respectively), those with both tumor cells and TIIC expressing PD-L1 a significantly worse DFS (p = 0.030). PD-L1 expression is present in 17% and 20% of salivary gland carcinoma cells and TIIC. Ten percent of the patient showed a PD-L1 positivity in both tumor cells and TIIC. This is related to high tumor grading and therefore might be a negative prognostic factor.

Concordance among four commercially available, validated programmed cell death ligand-1 assays in urothelial carcinoma

BackgroundAntibodies targeting the programmed cell death-1 (PD-1)/PD-ligand 1 (PD-1/PD-L1) checkpoint have shown promising clinical activity in patients with advanced urothelial carcinoma (UC). Expression of PD-L1 in UC tumors has been investigated using different antibody clones, staining protocols, and scoring algorithms. The aim was to establish the extent of concordance among PD-L1 immunohistochemistry (IHC) assays.MethodsTumor biopsy samples (N = 335) were assessed using four commercially available PD-L1 assays: VENTANA SP263, VENTANA SP142, PD-L1 IHC 28–8 pharmDx, and PD-L1 IHC 22C3 pharmDx. PD-L1 analytical staining and classification concordance, including agreement between clinically relevant scoring algorithms, were investigated using overall/positive/negative percentage agreement (OPA/PPA/NPA).ResultsGood analytical correlation was observed among the VENTANA SP263, PD-L1 IHC 22C3 pharmDx, and PD-L1 IHC 28–8 pharmDx assays for tumor cell (TC) and immune cell (IC) PD-L1 staining with Spearman rank coefficients of 0.92–0.93 for TCs and 0.88–0.91 for ICs. However, concordance (preset criterion: ≥85%) between patient PD-L1 status when applying the TC or ICICArea ≥ 25% (VENTANA SP263) cutoff was only achieved for PD-L1 IHC 22C3 pharmDx versus VENTANA SP263 (OPA 92.2%, PPA 86.4%, NPA 95.4%). Differences were observed between patient populations with UC tumors classified as PD-L1 high versus PD-L1 low/negative using combined positive score (CPS) ≥1, CPS ≥10, IC ≥5%, and TC/IC ≥25%.ConclusionsThe VENTANA SP263 and PD-L1 IHC 22C3 pharmDx assays are analytically similar in UC. When the different PD-L1 assays were combined with their specified clinical scoring algorithms, differences were seen in patient classification driven by substantial differences in scoring approaches.

JAK2/PD-L1/PD-L2 (9p24.1) amplifications in renal cell carcinomas with sarcomatoid transformation: implications for clinical management

Amplifications of JAK2, PD-L1, and PD-L2 at 9p24.1 lead to constitutive expression of PD-L1. This, coupled with JAK2-activation dependent upregulation of PD-L1 and adaptive/induced expression leads to higher tumor PD-L1 expression and immune evasion. Renal tumors were therefore evaluated for 9p24.1 amplifications. A combination of next generation sequencing-based copy number analysis, fluorescence in situ hybridization for JAK2/INSL6 and PD-L1/PD-L2 and immunohistochemistry for phospho-STAT3 (downstream target of JAK2), PD-L1, PD-L2, and PD-1 was performed. In this study we interrogated a “Discovery” cohort of 593 renal tumors, a “Validation” cohort of 398 high-grade renal tumors, The Cancer Genome Atlas (879 cases) and other public datasets (846 cases). 9p24.1 amplifications were significantly enriched in renal tumors with sarcomatoid transformation (5.95%, 15/252) when compared to all histologic subtypes in the combined “Discovery”, “Validation” and public datasets (16/2636, 0.6%, p < 0.00001). Specifically, 9p24.1 amplifications amongst sarcomatoid tumors in public datasets, the “Discovery” and “Validation” cohorts were 7.7% (6/92), 15.1% (5/33), and 3.1% (4/127), respectively. Herein, we describe 13 cases and amplification status for these was characterized using next generation sequencing (n = 9) and/or fluorescence in situ hybridization (n = 10). Correlation with PD-L1 immunohistochemistry (n = 10) revealed constitutive expression (mean H-score: 222/300, n = 10). Analysis of outcomes based on PD-L1 expression in tumor cells performed on 282 cases (“Validation” cohort) did not reveal a significant prognostic effect and was likely reflective of advanced disease. A high incidence of constitutive PD-L1 expression in tumor cells in the “Validation” cohort (H-Score ≥250/300) was noted amongst 83 rhabdoid (6%) and 127 sarcomatoid renal tumors (7.1%). This suggests additional mechanisms of constitutive expression other than amplification events. Importantly, two patients with 9p24.1-amplified sarcomatoid renal tumors showed significant response to immunotherapy. In summary, a subset of renal tumors with sarcomatoid transformation exhibits constitutive PD-L1 overexpression and these patients should be evaluated for enhanced response to immunotherapy.

Next Generation Sequencing and PD-L1 Immunohistochemistry in Pleural Fluids

Next Generation Sequencing and PD-L1 Immunohistochemistry in Pleural Fluids

Liquid biopsy in the era of immuno-oncology: is it ready for prime-time use for cancer patients?

The emergence of immunotherapy in oncology requires the discovery, validation and subsequent adoption of robust, sensitive and specific predictive and prognostic biomarkers for daily practice. Until now, anti-PD-L1 immunohistochemistry (IHC) on tissue sections has been the only validated companion diagnostic test for first-line immunotherapy for advanced and metastatic cancer, notably non-small-cell lung cancer (NSCLC). However, detection of this biomarker presents limitations that have stimulated the development of other biomarkers and other approaches. Within this context, the use of a liquid biopsy (LB) could provide an important complementary or alternative added value to PD-L1 IHC. In this review, we discuss how LBs have been used in the field of immuno-oncology (I-O) to predict response, relapse or adverse advents for patients undergoing immune-checkpoint inhibitor (ICI) therapy (anti-PD-1/PD-L1 and CTLA-4) and we highlight recent developments. Circulating tumor cells (CTCs), cell-free DNA (cfDNA), proteins and cytokines detected in plasma as well as circulating T-lymphocytes are discussed as potential sources for developing new I-O biomarkers. The quantification of cfDNA as a predictive biomarker, as well as its sequencing for the determination of tumor mutational burden, is already well advanced. Additionally, the quantification of PD-L1 from CTCs, bound on exosomes or free in plasma, as well as the determination of cytokines, are also being actively investigated with promising results having recently been published. Lastly, analysis of T-lymphocytes, especially by analyzing the T-cell receptor, has recently emerged as a valuable biomarker that might become relevant for the prediction of response to ICIs. While LBs have not yet been implemented in routine I-O clinical practice, recent promising data and rapidly advancing technologies indicate that this approach has the potential to soon personalize the clinical management of cancer patients receiving ICIs.

Correlation of PD-L1 Expression Tested by 22C3 and SP263 in Non-Small Cell Lung Cancer and Its Prognostic Effect on EGFR Mutation-Positive Lung Adenocarcinoma.

BackgroundProgrammed death-ligand 1 (PD-L1) expression is tested by immunohistochemistry (IHC)—22C3, SP263, and SP142. The aim of this study is to evaluate the correlation among the three methods of PD-L1 IHC in non-small cell lung cancer (NSCLC) and clinical significance of PD-L1 expression in lung adenocarcinoma with an epidermal growth factor receptor (EGFR)–tyrosine kinase domain mutation.MethodsThe results of 230 patients who were pathologically confirmed as having NSCLC; tested using PD-L1 IHC 22C3, SP263, and SP142 methods; and evaluated via the peptide nucleic acid clamping method to confirm EGFR mutation, were analyzed in this study.Results164 patients underwent both the SP263 and 22C3 tests. There was a significant positive correlation between the outcomes of the two tests (Spearman correlation coefficient=0.912, p<0.001), with a derived regression equation as follows: 22C3=15.2+0.884×SP263 (R2=0.792, p<0.001). There was no relationship between the expression of PD-L1 and clinical parameters, including EGFR–tyrosine kinase inhibitor (TKI) mutation. The PD-L1 expression in patients treated with EGFR-TKI yielded a 2-month-shorter progression period than that in the PD-L1–negative group. However, this did not reach statistical significance (PD-L1<1% vs. PD-L1≥1%, 10 months vs. 8 months).ConclusionThe results of the 22C3 and those of SP263 methods were in good correlation with one another. Since the PD-L1 expression is not influenced by the EGFR mutation, it is necessary to perform a PD-L1 test to set the treatment direction in the patients with EGFR-mutant NSCLC.

Tumor mutational burden.

Tumor mutational burden.

Tumor-draining lymph nodes demonstrate a suppressive immunophenotype in patients with non-small cell lung cancer assessed by endobronchial ultrasound-guided transbronchial needle aspiration: A pilot study

ObjectivesTumor draining lymph nodes (TDLN) are key sites of early immunoediting in patients with non-small cell lung cancer (NSCLC) and play an important role in generating anti-tumor immunity. Immune suppression in the tumor microenvironment has prognostic implications and may predict therapeutic response. T cell composition of draining lymph nodes may reflect an immunophenotype with similar prognostic potential which could be measured during standard-of-care bronchoscopic assessment. In this study, we compared the immunophenotype from different sites within individuals to primary tumor characteristics in patients with NSCLC to see whether there were tumor-regional differences in immunophenotype which could be evaluated from transbronchial needle aspirates. Materials and MethodsTwenty patients were enrolled in this study and had tissue (lymph node aspirates and/or peripheral blood) obtained during standard of care bronchoscopy with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for diagnosis or staging of known or suspected NSCLC. Aspirates and blood underwent flow-assisted cell sorting and a subset of sorted effector T cells underwent RNA quantitation to determine feasibility of this approach. Immunophenotypic patterns from twelve patients with paired data from tumor-draining and non-tumor draining lymph nodes (NDLN) were compared relative to one another and based on PD-L1 immunohistochemistry and primary tumor histology. ResultsTDLN had significantly fewer CD4+ T cells (12.68% vs 27%, p = 0.002) and significantly more regulatory T cells (Treg, 12.03% vs 9.52%, p = 0.03) relative to paired NDLN suggesting tumor-regional immunosuppression. There were significantly more Treg in NDLN relative to paired PBMC (9.52% vs 5.6%, p = 0.016). Patients with PD-L1 expression ≥50% had significantly greater tumor-regional CD4+ T cell depletion compared to patients with PD-L1 expression <50% (−35.98% vs −1.89%, p = 0.0357; negative values represent absolute difference between paired TDLN and NDLN). ConclusionsIn patients with NSCLC, TDLN have a suppressive immunophenotype correlating with tumor PD-L1 status and can be assessed during routine EBUS-TBNA.

Abstract CT228: Phase Ib clinical study of CBP501, cisplatin and nivolumab administered every 3 weeks in patients with advanced refractory tumors. Dose escalation cohort

Abstract Background: CBP501 is a 12 amino acid calmodulin-modulating peptide that increases platinum influx into tumor cells and induces tumor immunogenic cell death. CBP501 also suppresses platinum-induced release of cytokines, such as IL-6, IL-10 and VEGF, lowers cancer stem cell populations and reduces the migration, invasion and epithelial-mesenchymal transition of tumor cells. Preclinical studies show synergistic anti-tumor activity when combined with platinum agents and immune checkpoint inhibitors (anti-PD1, PD-L1 and CTLA-4 antibodies) in the CT26 syngeneic mouse model. Here, we report dose-finding results from a Phase Ib study of CBP501 combined with cisplatin and nivolumab in a triple drug combination. Methods: An open-label phase I trial was conducted using a 3+3 design: CBP501 and cisplatin were dosed simultaneously by 1h infusion Q3W at 4 different combined dose levels (CBP501: 16 or 25 mg/m2; cisplatin: 60 or 75 mg/m2). Nivolumab (240 mg) was dosed on the same day, as a 1h infusion following CBP501/cisplatin. The DLT observation period was during the first 21-day cycle. Immuno-histochemistry for PD-L1 and CD8-positivity were performed in archival tumor samples. Results: As of 28 Dec. 2018 for AEs, 19 patients were dosed and no DLTs were reported. Treatment-related adverse events as assessed by the investigator occurred in 19 patients (100%). Fifteen patients experienced infusion reactions related to CBP501, manifesting as erythema or hives. These were managed with interruption of the infusion, administration of antihistamines, and resumption of the infusion if reactions resolved within 30 minutes (17/19 treated patients). These reactions occurred at both CBP501 dose levels evaluated. Other events occurring in &amp;gt;/= 2 patients included anemia (10), fatigue/asthenia (8), leukopenia/neutropenia/granulocytopenia (7), thrombocytopenia (5), anorexia/weight loss (4), nausea (3), vomiting (2), hypersensitivity (2) lymphopenia (2), and acute kidney injury/renal failure (2). Eleven treatment-related Grade 3 or 4 AEs occurred in six patients, anemia (4), acute renal failure (1), diarrhea (1), fatigue (1), acute encephalopathy (1), hypocalcemia (1), neutropenia/leukopenia (1), and thrombocytopenia (1). The recommended phase 2 dose (RP2D) is 60 mg/m2 cisplatin with 25 mg/m2 CBP501 and 240 mg nivolumab. Partial responses were seen for 2 heavily pretreated patients, 1 with pancreatic cancer (PD-L1 (+) tumor cells 70%, CD8 (+) cells 18%) and 1 with microsatellite stable (MSS) colorectal cancer. Disease control rate (DCR), defined as CR, PR and &amp;gt;3M SD was 35% (6 /17 evaluable patients). Notably, DCR was 50% in heavily pretreated pancreatic cancer (2/4) and cholangiocarcinoma (1/2) patients. Conclusions: The triple-drug combination is tolerable and an RP2D has been determined. Pancreatic and MSS colorectal cancers were selected for expansion cohorts. Pre- and on-treatment biopsies will be obtained to assess modulation of the immune microenvironment by combination treatment. (NCT03113188). Citation Format: Marc Matrana, Frank Tsai, Suma Satti, James Cleary, Jacob Estes, Khanh Do, Valentin Kolmakov, Takumi Kawabe, Geoffrey I. Shapiro. Phase Ib clinical study of CBP501, cisplatin and nivolumab administered every 3 weeks in patients with advanced refractory tumors. Dose escalation cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT228.