Programmed Death-ligand 1 Immunostaining Research Articles

Feasibility of Endobronchial Ultrasound Guided Miniforceps Biopsy for PD-L1 Expression and Quantitative Tissue Analysis.

Testing for targeting programmed death ligand 1 (PD-L1) is standard of care for patients with newly diagnosed non-small cell lung cancer (NSCLC) but is only approved for use with core biopsy specimens. Endobronchial ultrasound guided miniforceps biopsy (EBUS-MFB) is an approach to obtain core biopsy material but data assessing the ability of EBUS-MFB to adequately test for PD-L1 is lacking. We evaluate the feasibility of EBUS-MFB to acquire adequate tissue for PD-L1 testing and look to compare the quality of specimens between EBUS-MFB and endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) using a standard method of tissue analysis. Twenty patients with suspected non-small cell lung cancer undergoing bronchoscopy were recruited for enrollment. For each patient with NSCLC diagnosed on rapid onsite pathology with EBUS-TBNA, EBUS-MFB was performed. PD-L1 immunostaining was completed to assess for adequacy. A comparison of tissue collection was performed using the total surface area measured by digital imaging. Among 20 patients, 65% were male with a mean age of 66 years with a total procedure time of 50 min and an average of 14 biopsy passes per procedure. 15 (75%) patients were diagnosed with NSCLC, and PD-L1 analysis was successfully performed in 12 of the 15 (80%). The mean total tissue area obtained by the MFB technique was 9.757 mm2 compared to 6.941 mm2 with TBNA (p = 0.427). In this feasibility study, EBUS-MFB was successful in performing PD-L1 testing in 80% of patients with NSCLC.

Three-dimensional iodine mapping quantified by dual-energy CT for predicting programmed death-ligand 1 expression in invasive pulmonary adenocarcinoma

We examined the association between texture features using three-dimensional (3D) io-dine density histogram on delayed phase of dual-energy CT (DECT) and expression of programmed death-ligand 1 (PD-L1) using immunostaining methods in non-small cell lung cancer. Consecutive 37 patients were scanned by DECT. Unenhanced and enhanced (3 min delay) images were obtained. 3D texture analysis was performed for each nodule to obtain 7 features (max, min, median, mean, standard deviation, skewness, and kurtosis) from iodine density mapping and extracellular volume (ECV). A pathologist evaluated a tumor proportion score (TPS, %) using PD-L1 immunostaining: PD-L1 high (TPS ≥ 50%) and low or negative expression (TPS < 50%). Associations between PD-L1 expression and each 8 parameter were evaluated using logistic regression analysis. The multivariate logistic regression analysis revealed that skewness and ECV were independent indicators associated with high PD-L1 expression (skewness: odds ratio [OR] 7.1 [95% CI 1.1, 45.6], p = 0.039; ECV: OR 6.6 [95% CI 1.1, 38.4], p = 0.037). In the receiver-operating characteristic analysis, the area under the curve of the combination of skewness and ECV was 0.83 (95% CI 0.67, 0.93) with sensitivity of 64% and specificity of 96%. Skewness from 3D iodine density histogram and ECV on dual energy CT were significant factors for predicting PD-L1 expression.

Update regarding the role of PD-L1 in oncocytic thyroid lesions on cytological samples

AimsSeveral papers have shown that programmed death-ligand 1 (PD-L1) expression is a relevant predictive biomarker in anti-PD-L1 cancer immunotherapy. While its role in several human cancers is correlated with poor...

Assessment of the RANTES Level Correlation and Selected Inflammatory and Pro-Angiogenic Molecules Evaluation of Their Influence on CRC Clinical Features: A Preliminary Observational Study.

Background and Objectives: Assessment of RANTES level and concentrations of inflammatory cytokines: programmed death ligand 1 (PD-L1), interferon gamma IFN-γ, tumor necrosis factor alpha (TNF-α), transforming growht factor β (TGF-β) (and angiogenesis factors: vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor C (VEGF C) in tumor and margin tissues of colorectal cancer (CRC,) and evaluation of RANTES influence on histopathological parameters (microvessel density (MVD), budding, tumor-infiltrating lymphocytes (TILs)), in relation to patients’ clinical features. Materials and Methods: The study used 49 samples of tumor and margin tissues derived from CRC patients. To determinate the concentration of RANTES, PD-L1, IFN-γ, TNF-α, TGF-β, VEGF-A, and VEGF-C, we used the commercially available enzyme-linked immunosorbent assay kit. Additionally, RANTES and PD-L1 expression was assessed with the use of IHC staining in both tumor cells and TILS in randomly selected cases. MVD was assessed on CD34-stained specimens. The MVD and budding were assessed using a light microscope. Results: We found significantly higher levels of RANTES, PD-L1, IFN-γ, TNF-α, TGF-β, VEGF-A, and VEGF-C in the tumor in comparison with the margin. The RANTES tumor levels correlated significantly with those of PD-L1, TNF-α, TGF-β, VEGF-A, and VEGF-C. The RANTES margin levels were significantly associated with the margin levels of all proteins investigated—PD-L1, IFN-γ, TNF-α, TGF-β, VEGF-A, and VEGF-C. Additionally, we observed RANTES- and PD-L1-positive immunostaining in TILs. In a group of 24 specimens, 6 different CRC tumors were positive for RANTES and PD-L1 immunostaining. The IFN-gamma concentration in both tumor and margin and TGF-β in tumor correlated with TILs. TILs were negatively associated with the patients’ disease stage and N parameter. Conclusions: RANTES activity might be associated with angiogenesis, lymphogenesis, and immune escape in CRC. RANTES is an important chemokine that is a part of the chemokine–cytokine network involved in the modulation of TME composition in CRC. Further research may verify which processes are responsible for the associations observed in the study.

False-negative programmed death-ligand 1 immunostaining in ethanol-fixed endobronchial ultrasound-guided transbronchial needle aspiration specimens of non-small-cell lung cancer patients.

AimsProgrammed death‐ligand 1 (PD‐L1) immunostaining is used to predict which non‐small‐cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD‐L1 inhibitors. PD‐L1 immunostaining is sometimes performed on alcohol‐fixed cytological specimens instead of on formalin‐fixed paraffin‐embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) results in diminished PD‐L1 immunostaining as compared with formalin fixation.Methods and resultsFFPE cell blocks from EBUS‐TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD‐L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory‐developed test (LDT). PD‐L1 positivity was determined with two cut‐offs (1% and 50%). Concordance of PD‐L1 positivity between the formalin‐fixed (gold standard) and ethanol‐prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol‐prefixed specimens showed false‐negative results at the 1% and 50% cut‐offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol‐prefixed specimens showed false‐negative results at the 1% cut‐off (kappa 0.67). At the 50% cut‐off, concordance was higher (kappa 0.91), with 12% of the ethanol‐prefixed specimens showing false‐negative results.ConclusionEthanol fixation of EBUS‐TBNA specimens prior to formalin fixation can result in a considerable number of false‐negative PD‐L1 immunostaining results when a 1% cut‐off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut‐off when immunostaining is performed with the 22C3 LDT.

PD-L1 Expression in Muscle Invasive Urothelial Carcinomas as Assessed via Immunohistochemistry: Correlations with Specific Clinical and Pathological Features, with Emphasis on Prognosis after Radical Cystectomy.

In the present study, we analyzed Programmed Death Ligand-1 (PD-L1) expression in radical cystectomy (RC) specimens from patients with muscle-invasive urothelial carcinoma (UC), in order to assess any correlations with specific clinicopathological features and its potential prognostic value. A multi-institutional study was performed within the departments of urology and pathology at the Mureș County Hospital, Romania, and Centre Hospitalier Lyon Sud, France. Sixty-nine patients with MIBC were included, for whom tumor histology (conventional versus histological variant/differentiation), tumor extension (T), lymph node involvement (N), and distant metastases (M) were recorded. PD-L1 immunostaining was performed using the 22C3 clone and was interpreted using the combined positive score (CPS) as recommended (Dako Agilent, Santa Clara, CA, USA). Positive PD-L1 immunostaining was more prevalent among UCs with squamous differentiation compared to conventional UCs and trended towards an improved OS (p = 0.366). We found the T stage to be a risk factor for poor survival in PD-L1-positive patients (HR 2.9, p = 0.021), along with the N stage in PD-L1-negative patients (HR 1.98, p = 0.007). No other clinicopathological factor was found to be significantly associated with PD-L1 positivity. Thus, we confirm the need for PD-L1 immunostaining prior to initiating immune checkpoint inhibitor therapy for a more accurate assessment of the patients’ chances of responding to treatment.

PD-L1 Expression in Normal Endocrine Tissues Is Not Increased Despite High Incidence of PD-1 Inhibitor-Associated Endocrinopathies

ObjectiveTreatment with immune-checkpoint inhibitors often results in endocrine immune-related adverse events (irAEs), affecting the pituitary, thyroid, adrenal, and parathyroid glands and pancreas. The mechanism underlying the endocrine irAEs has not been fully elucidated, and it remains unclear why endocrine organs are so commonly affected. In the present study, we evaluated immunostaining patterns of programmed death-ligand 1 (PD-L1) in normal endocrine tissues to determine whether increased expression may explain the predilection of endocrinopathies in patients treated with programmed cell death-1 inhibitors. MethodsNormal formalin-fixed paraffin-embedded endocrine tissues (pituitary, thyroid, adrenal, pancreas, and parathyroid) were collected from our hospital’s pathology tissue archive. The tissues were assessed for membranous and cytoplasmic PD-L1 immunostaining using the Dako 22C3 pharmDx assay on an automated staining platform. ResultsWe examined 49 endocrine tissues, including 12 thyroid, 5 pancreatic, 17 adrenal, 5 parathyroid, and 10 pituitary samples. Samples with less than 1% membranous PD-L1–positive cells were considered negative, while those with more than 1% of PD-L1 membranous staining were considered positive. Immunostaining result of immune-related cells was also evaluated, considering the cytoplasmic PD-L1–positive cells with the same cutoff of 1%. None of the endocrine tissues demonstrated PD-L1 positivity higher than 1% in the relevant cells. ConclusionWhile our results do not suggest a role of PD-L1 expression in the pathogenesis of endocrine irAEs, they may serve as a basis for future studies further investigating the mechanisms of autoimmune, inflammatory, or malignant endocrine conditions.

Formalin fixation for optimal concordance of programmed death-ligand 1 immunostaining between cytologic and histologic specimens from patients with non-small cell lung cancer.

BackgroundImmunohistochemical staining of programmed death‐ligand 1 (PD‐L1) is used to determine which patients with non–small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD‐L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD‐L1 immunoreactivity was studied.MethodsPaired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine‐needle aspiration on pneumonectomy/lobectomy specimens. Formalin‐fixed, agar‐based or CytoLyt/PreservCyt‐fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory‐developed test) antibodies. PD‐L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD‐L1–expressing human cell lines fixed in various fixatives at increasing increments in fixation duration.ResultsAgar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair‐to‐moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol‐based fixatives showed less PD‐L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory‐developed test and the 22C3 pharmDx assay.ConclusionsPerforming PD‐L1 staining on cytologic specimens fixed in alcohol‐based fixatives could result in false‐negative immunostaining results, whereas fixation in formalin leads to higher and more histology‐concordant PD‐L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin.

Prognostic relevance of programmed cell death protein 1/programmed death-ligand 1 pathway in thymic malignancies with combined immunohistochemical and biomolecular approach

ABSTRACT Background The aim of the study was to investigate Programmed cell Death protein 1 (PD-1) and Programmed Death-Ligand 1 (PD-L1) and their mRNA expression in thymic epithelial tumors (TETs). Research design and methods We analyzed 68 samples of formalin-fixed paraffin-embedded tissue (63 thymomas and 5 thymic carcinomas). PD-1 and PD-L1 protein expression were evaluated by immunohistochemistry, and mRNA expression was evaluated by real-time PCR. Results M/F ratio was 33/35, and median age was 60.5 years. Twenty patients had Myasthenia Gravis (MG). In the subgroup with large tumors (>5 cm), PD-L1 mRNA overexpression was significantly associated with worse prognosis vs. patients with no mRNA overexpression (p = 0.0083) and simultaneous PD-L1 immunostaining (>1%); PD-L1 mRNA overexpression was significantly associated with worse prognosis, respect to patient with PD-L1 negative immunostaining, and no PD-L1 mRNA overexpression (p = 0.0178). The elderly patients (>60 years) with large tumors showed worse prognosis (p = 0.0395). PD-L1 immunostaining (>50%) resulted to be significantly associated with MG. Conclusions Our data suggest the potential involvement of the PD-1 and PD-L1 pathway in TETs’ progression. According to our results, it may be helpful to design future trials with anti-PD-1 drugs to establish high-risk patients after surgery.

Expression of programmed death-ligand 1 (PD-L1) in human pituitary neuroendocrine tumor.

To explore the programmed death-ligand 1 (PD-L1) expression in varied subtypes of pituitary neuroendocrine tumors with assessment of their clinical behavior at diagnosis and follow-up. We conducted a retrospective monocentric study, including all patients operated in the Academic Hospital of Angers (France) for a pituitary neuroendocrine tumor between 2012 and 2018. PDL-1 immunostaining was performed using a European Conformity-In Vitro Diagnostic-labeled anti-PDL1 antibody (clone 22C3). PD-L1 immunostaining was evaluated as the percentage of tumor cells showing positive membrane staining, into four grades: grade 0 = < 1%, grade 1 = 1 to 5%, grade 2 = 6 to 49% and grade 3 = ≥ 50%. PD-L1 expression was compared with tumor features (secretion, proliferation, invasion) and outcome. The study included 139 pituitary neuroendocrine tumors, including 84 (60%) nonfunctioning adenomas. Twenty-five pituitary neuroendocrine tumors were PD-L1 positive (18%), including 3 grade 3, 8 grade 2 and 14 grade 1. PD-L1 expression was not different between functioning and nonfunctioning adenomas (p = 0.26). Among 16 tumors with proliferative markers (Ki-67 ≥ 3% and p53 positive), only one was PD-L1 positive. In our series, PD-L1 was expressed in a rather small proportion of PitNET (18%), and this immune marker was not associated with any biological characteristic or behavior of the pituitary tumors. Thus, PD-L1 staining may be necessary before considering PD-L1 blockage in pituitary neuroendocrine tumors, in case of therapeutic impasse.

RAD51Bme as predictive biomarker for PD-1 blockade response in non-small cell lung cancer.

e15235 Background: Lung cancer (LC) cells frequently express high levels of programmed death-ligand 1 (PD-L1). Although these levels grossly correlate with likelihood of response to specific checkpoint inhibitors, prediction of response is rather imperfect and, thus, more accurate predictive biomarkers are mandatory. We examined the methylation profile of RAD51B, a DNA-repair gene, as candidate predictive biomarker for efficacy of anti-PD-1 therapy in patients with non-small cell lung cancer (NSCLC), correlating with patients’ outcome. Methods: PD-L1 immunoexpression and RAD51Bme levels were analysed in samples of NSCLC obtained from patients not treated with anti-PD-1 blockade (testing cohort) and patients treated with anti-PD-1 (validation cohort). Results: Of a total of 127 patients assessed, 58.3% depicted positivity for PD-L1 (PD-L1+). RAD51Bme levels significantly associated with PD-L1 immunoexpression. Patients with clinical benefit from immunotherapy disclosed higher RAD51Bme levels (p = 0.04) and significantly higher median progression free survival (PFS) (p = 0.02). PD-L1+ was also associated with longer overall survival (p = 0.03). Conclusions: We demonstrated that RAD51Bme levels might be combined with validated predictive biomarker PD-L1 immunostaining to select patients who will most likely experience clinical benefit from PD-1 blockade. The predictive value of RAD51Bme should be confirmed in prospective studies.

MON-287 Expression of Programmed Death-Ligand 1 (PD-L1) in Human Pituitary Neuroendocrine Tumor

IntroductionSome Pituitary NeuroEndocrine Tumors (PitNET) present an aggressive evolution and are resistant to standard management. Immunotherapy have shown durable efficacy in a variety of malignancies. The aim of this study was to explore the programmed death-ligand 1 (PD-L1) expression in varied subtypes of pituitary adenomas with assessment of their clinical behavior at diagnosis and follow-up.MethodsWe conducted a retrospective monocentric study, including all patients operated a PitNET between 2012 and 2018. PDL-1 immunostaining were performed using an European Conformity-In-Vitro-Diagnostic labeled anti-PDL1 antibody (Clone 22C3). PD-L1 immunostaining was evaluated as the percentage of tumor cell showing positive membrane staining, into four grades: grade 0 = <1%, grade 1 = 1 to 5%, grade 2 = 6 to 49% and grade 3 = ≥ 50%. PD-L1 expression was compared with tumor features (secretion, proliferation, invasion) and outcome.ResultsThe study included one hundred and thirty-nine PitNET, including 84 (60%) nonfunctioning adenomas. Twenty-five PitNET were PD-L1 positive (18%), including 3 grade 3, 8 grade 2 and 14 grade 1. PD-L1 expression was not different between functioning and non-functioning adenomas (p=0.26). Among sixteen tumors with proliferative markers (Ki-67 ≥ 3% and p53 positive), only one was PD-L1 positive.ConclusionIn our series, pituitary tumors rarely exhibit PD-L1 expression and this immune marker did not seem to be associated with any biological characteristic or behavior of the pituitary tumors. Thus, PD-L1 staining is necessary before considering PD-L1 blockage in PitNET, in case of therapeutic impasse.

RAD51Bme Levels as a Potential Predictive Biomarker for PD-1 Blockade Response in Non-Small Cell Lung Cancer.

Lung cancer (LC) cells frequently express high levels of programmed death-ligand 1 (PD-L1). Although these levels grossly correlate with the likelihood of response to specific checkpoint inhibitors, the response prediction is rather imperfect, and more accurate predictive biomarkers are mandatory. We examined the methylation profile of RAD51B (RAD51Bme) as a candidate predictive biomarker for anti-PD-1 therapy efficacy in non-small cell lung cancer (NSCLC), correlating with patients’ outcome. PD-L1 immunoexpression and RAD51Bme levels were analysed in NSCLC samples obtained from patients not treated with anti-PD-1 (Untreated Cohort (#1)) and patients treated with PD-1 blockade (Treated Cohort (#2)). Of a total of 127 patients assessed, 58.3% depicted PD-L1 positivity (PD-L1+). RAD51Bme levels were significantly associated with PD-L1 immunoexpression. Patients with PD-1 blockade clinical benefit disclosed higher RAD51Bme levels (p = 0.0390) and significantly lower risk of disease progression (HR 0.37; 95% CI: 0.15–0.88; p = 0.025). Combining RAD51Bme+ with PD-L1+ improved the sensitivity of the test to predict immunotherapy response. PD-L1+ was also associated with lower risk of death (HR 0.35; 95% CI: 0.15–0.81; p = 0.014). Thus, RAD51Bme levels might be combined with validated predictive biomarker PD-L1 immunostaining to select patients who will most likely experience clinical benefit from PD-1 blockade. The predictive value of RAD51Bme should be confirmed in prospective studies.

PD-L1 and thyroid cytology: A possible diagnostic and prognostic marker.

Programmed death-ligand 1 (PD-L1) expression is emerging as an important predictive biomarker in anti-PD-L1 cancer immunotherapy. Its role has been clearly defined in various human cancers and is linked to a poor prognosis and resistance to anticancer therapies. The role of PD-L1 in thyroid cancers has not been well defined in fine-needle aspiration cytology (FNAC). The authors examined the performance of PD-L1 immunostaining in liquid-based cytology (LBC) to determine whether it could be a biomarker of malignancy or aggressive disease. From January 2018 to March 2019, 236 thyroid lesions, which had been diagnosed by FNAC as indeterminate lesions, suspicious for malignancy (SFM), and malignant, were enrolled. PD-L1 immunostaining was performed on both LBC and corresponding histology samples. The FNAC cohort included 50 benign negative controls, 42 samples of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), 33 samples of follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 53 samples that were suspicious for malignancy (SFM), and 58 malignant samples. AUS/FLUS samples included 3 goiters, 32 follicular adenomas (FAs), 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 5 invasive follicular variants of papillary thyroid carcinoma (I-FVPTCs), and 1 follicular carcinoma; whereas FN/SFN samples included 24 FAs and 9 malignancies (4 I-FVPTCs, 1 NIFTP, 3 papillary thyroid carcinomas [PTCs], and 1 oncocytic follicular carcinoma). The 53 SFM samples were diagnosed on histopathology as 2 FAs, 5 NIFTPs, 15 I-FVPTCs, and 31 PTCs; whereas the 58 malignant specimens included 5 NIFTPs, 5 I-FVPTCs, and 48 PTCs. Increased plasma membrane and cytoplasmic PD-L1 expression was found in 79 cases (38.5%), including 61 PTCs (conventional and variants). Negative PD-L1 expression was found in NIFTPs and FAs. A BRAF V600E mutation was identified in 15% of PD-L1-positive malignancies. The current data suggest that PD-L1 expression in the thyroid gland might represent a marker of malignancy that correlates with PTC, but not with NIFTP. Thyroid neoplasms with PD-L1 expression also ae enriched with BRAF V600E mutations, suggesting that they are associated with more aggressive behavior.

Programmed death-ligand 1 expression on direct Pap-stained cytology smears from non-small cell lung cancer: Comparison with cell blocks and surgical resection specimens.

Programmed death-ligand 1 (PD-L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy. The current study evaluated the feasibility and efficacy of PD-L1 immunostaining and quantitation on direct Papanicolaou-stained cytological smears compared with formalin-fixed paraffin-embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona). PD-L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC). In 57 sets of samples, both PD-L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin-fixed paraffin-embedded samples was observed in 97.3% of the cases. The quantification of PD-L1 expression on direct Papanicolaou-stained cytology smears is feasible and reliable for both PD-L1 assays.

Abstract P4-02-10: Are fine-needle aspiration (FNA)-derived cell blocks a useful tissue sample surrogate for testing conventional biomarkers and PD-L1 in breast cancer?

Abstract Background The diagnosis of breast cancer (BC) is based on clinical examination in combination with imaging and confirmed by pathological assessment of core needle biopsy (CNB) or fine needle aspiration (FNA). The biological profile of the lesion is needed to define prognosis and guide therapy. Given the importance of an early and minimally invasive diagnosis, we aimed to verify whether the biological features detected in FNA-derived cytological material reflect the biological characteristics of surgical specimens. Methods We used immunohistochemistry and fluorescence in situ hybridization (FISH) to study a panel of biomarkers (ER, PgR, Ki67 and HER2 in 93 patients, programmed death-ligand 1 (PD-L1) in 20 patients) in FNA-derived cell blocks of BC, comparing the results with those obtained on the histological specimens.Immunostaining was performed with the Ventana Benchmark XT system and the Ultraview DAB Detection Kit (Ventana Medical Systems). Confirm anti-ER (clone SP1, Ventana), Confirm Anti-PgR (clone 1E2, Ventana) and Ki67 (clone Mib-1, Dako, Carpinteria, CA, US) antibodies were used. Ventana PD-L1 (SP263) assay (Ventana Medical Systems) was used for PD-L1 immunostaining. HER2 status was analyzed by FISH using PathVysion kit (Abbott Molecular, Abbott Park, Illinois, IL, USA). Results Median immunopositive values of ER, PgR Ki67, and PD-L1 were similar in cell blocks and surgical samples. Concordance for ER and PgR between FNA and histological samples was 98% and 84%, respectively. With regard to Ki67 and HER2 status, concordance between the two specimen types was 90% and 96%, respectively. PD-L1 expression analyzed in FNA-derived samples was 100% concordant with that of surgical specimens. Tumor subtype classification for triple-negative and HER2-positive tumors in FNA samples was always concordant with the subtype determined in surgical material. Table 1.Concordance of tumor subtype classification between FNAB-derived and histological samples.Overall series (n=93)87%Luminal A (n=26)81%Luminal B (n=47)86%HER2-positive (n=6)100%Triple-negative (n=14)100% Conclusions We showed that biological marker determination in FNA-derived cell blocks is feasible and provides useful information and comparable results with those obtained by histological evaluation. Given the low cost of the procedure and its minimal impact on patients, that cytological samples could be used as an alternative to tissue samples for early BC biomarker evaluation to facilitate the planning of tailored neoadjuvant therapy. Citation Format: Bravaccini S, Ravaioli S, Rocca A, Tumedei MM, Bucchi E, Malmesi M, Medri L, Bonafè M, Puccetti M. Are fine-needle aspiration (FNA)-derived cell blocks a useful tissue sample surrogate for testing conventional biomarkers and PD-L1 in breast cancer? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-02-10.

Validation of the immunohistochemical expression of programmed death ligand 1 (PD-L1) on cytological smears in advanced non small cell lung cancer

Introduction The assessment of PD-L1 expression by immunohistochemistry is mandatory for the administration as first-line therapy of the anti PD-1 check-point inhibitor Pembrolizumab in patients with advanced non-small-cell lung cancer (NSCLC). Currently, only formalin-fixed paraffin-embedded samples are acceptable for PD-L1 immunostaining with the anti-PD-L1 antibodies 22-C3 and SP263. We investigated retrospectively the accuracy of the anti PD-L1 antibodies 22-C3, 28-28, SP263 in 50 paired histological samples and cytological smears of NSCLC patients.Results The accuracy of the three antibodies for the detection of PD-L1 in histological samples was higher for the antibody SP263 (AUC/ROC = 1) compared to the clones 28-8 (AUC/ROC =,991) and 22-C3 (AUC/ROC =,942). The overall concordance between histological samples and cytological smears using the SP263 clone was moderate (kappa = 0,364). However when the cyto-histological concordance was calculated using just the <50% vs ≥50% cut-off the agreement (kappa = 0.626) was good. The accuracy of the antibody SP263 in cytological smears was good (AUC/ROC =,921). A fluorescent in situ hybridization analysis on 10 histological cases positive for PD-L1 at immunohistochemistry showed amplification of the CD274 gene only in one case.Conclusions Immunocytochemical staining for PD-L1 in diagnostic cytological smears of NSCLC is feasible and applicable at least using the >50% cancer cell cut-off. The three antibodies SP263, 22-C3 and 28-8 are all suitable for the diagnostic detection of PD-L1 on tissue sections with a superiority of the SP263 clone. The implementation of PD-L1 immunocytochemistry on cytological smears will likely expand the pool of NSCLC patients candidate to first-line immunotherapy.

PDL-1 and PDL1 expressions in clear cell renal cell carcinoma (ccRCC) of metastatic patients with sunitinib first-line treatment.

e14002 Background: Anti vascular growth epithelial factor (VEGF) therapies are currently used in first line of metastatic ccRCC, but some patients are inherently resistant to these treatments. Immunotherapy based on disruption of immune checkpoints, such as Programmed Death Ligand 1 (PD-L1) and Programmed Death 1 (PD-1), has been showed promising results. Interaction between PD-L1 and PD-1 down-regulates antitumor activity and could be involved in resistance to anti-VEGF therapies Methods: In this study, we assessed PD-L1 and PD-1 expressions by immunohistochemistry in primary ccRCC of metastatic patients with sunitinib first-line treatment. From 2 centers, 92 patients were restrospectively included and clinical data were collected with a median follow up of 22 months after sunitinib introduction. PD-L1 immunostaining was evaluated on the highest Fuhrman grade areas (n = 92) and PD-1 on the most inflammatory territory when the corresponding block was available (n = 47). PD-L1 and PD-1 were considered over...