Programmed Death-ligand 1 Staining Research Articles

A digital pathologist support tool for accurate detection of PD-L1 in head and neck squamous cell carcinoma.

e18031 Background: The programmed death-ligand 1 (PD-L1) (22C3) antibody is a companion diagnostic to the check-point inhibitor drug pembrolizumab for head and neck squamous cell carcinoma (HNSCC). Specifically, PD-L1 expression, as determined by a Combined Positive Score (CPS) ≥ 1, defines the clinical decision point for treatment. However, to calculate the CPS, pathologists must account for PD-L1+ tumor cells (TC) and immune cells (IC, i.e., lymphocytes, macrophages) – a cumbersome and time-consuming task that is prone to intra/inter-pathologist variability. We developed a digital image analysis (IA) based solution as a pathologist support tool that accurately and precisely scores images of PD-L1 stained HNSCC tissues. Here we compared the performance of the digital method with manual pathology. To address the literature on the differential regulation of PD-L1 by IC and TC in treatment response, we also examined how PD-L1+ IC contribute to the CPS as compared to PD-L1+ TC. Methods: 19 HNSCC samples were immunohistochemically stained for PD-L1 (22C3). Slides were assessed for CPS by manual pathology: CPS = (# PD-L1 staining cells [TC, lymphocytes, macrophages]/total # viable TC) x 100; wherein CPS < 1 (no PD-L1 expression; no treatment) and CPS ≥ 1 (PD-L1 expression; treatment). Slides were scanned at 20X and analyzed using proprietary IA solutions that identify and separate TC from IC and quantify their PD-L1 expression. Resulting IA markups of cell detection and PD-L1 expression were reviewed by an MD pathologist for accurate cell recognition and stain classification. Upon pathologist approval of the IA performance, PD-L1 staining was evaluated by digital IA for CPS, % PD-L1+ TC, and % PD-L1+ IC. Pearson’s correlation analyses were conducted to assess the concordance between manual and digital CPS along with the association between CPS and PD-L1+ IC versus PD-L1+ TC. Results: Digital and manual CPS were significantly correlated ( r = 0.76, p = 0.00016) and concordant in treatment binning for 14/19 samples; for the 5 discordant samples, manual CPS binned them for no treatment while digital CPS binned them for treatment. Digital CPS significantly correlated with the % PD-L1+ IC ( r = 0.90, p < 0.00001) along with the % PD-L1+ TC ( r = 0.98, p < 0.00001). All 6 samples binned for no treatment by digital CPS had < 1% PD-L1+ IC, whereas all but 2/13 samples binned for treatment by digital CPS had ≥ 1% PD-L1+ IC. Conclusions: The digital method for assessing PD-L1 expression in HNSCC performs similarly to manual pathology with improved accuracy for detecting and quantifying IC versus TC in the CPS calculation. Moreover, the % of PD-L1+ IC seems to contribute substantially to the CPS, to a similar degree as the % of PD-L1+ TC. Given the concordance with manual pathology, this digital method can support pathologists as a clinical diagnostic tool. As such, a clinical validation study of this digital method in a larger HNSCC cohort is underway.

Programmed death ligand-1 (PD-L1) expression in patients (pts) with metastatic renal cell carcinoma (mRCC) treated with nivolumab (NIVO) in combination with stereotactic body radiotherapy (SBRT) in NIVES study.

4558 Background: The NIVES study represents the first prospective trial with NIVO in combination with SBRT in pre-treated mRCC patients. This study did not meet the primary endpoint in terms of objective response rate (ORR) as previously reported. However this combination showed a faster time to treatment response, a long progression free survival and median duration of response without increasing toxicities. Here we have tested with an exploratory analysis the correlation between PD-L1 expression and clinical outcomes in pts treated with NIVO plus SBRT. Methods: PD-L1 expression was assessed in archival collected tumour samples in our central laboratory using 4 commercial kits for immunoistochemical (ICH) analysis (clone 22C3 pharm DX Dako Agilent, 28.8 Abcam and SP142 and SP263 Ventana Medical System). A tumor cell was considered positive if any membranous staining was found regardless of the intensity. In particular the immunostaining was scored 0 when all tumor cells were unstained (PD-L1-negative), 1+ when < 1% positive tumor cells were counted, 2+ when the percentage was between 1% and 50%,3+ when the number of stained cells was more than 50%. ORR and overall survival (OS) were correlated with PD-L1 staining. Results: Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 44 of 69 pts enrolled in the NIVES study. Twenty-two pts of 44 (50%) were considered PD-L1-negative using all the 4 commercial kits for ICH analysis, while 14 of 44 pts (31,8%) were defined PD-L1 weakly positive (positive tumor cells < 1% at least in one kit for ICH). Eight of 44 pts (18.1%) were defined PD-L1 strong positive when at least one kit for ICH scored 2+ or 3+. About the correlation between ORR and PDL1 staining in the 42 pts (2/44 pts are not evaluable for ORR), ORR was 18.2% (95% CI, 5.2% to 40.3%) in the PD-L1-negative group vs 20% (95% CI, 5.7% to 43.7%) in weakly/strongly PD-L1 positive (p = 1.00). Among the 44 pts in the intention-to-treat population with available PD-L1 status, median OS was not significantly different between pts with PD-L1 negative (20.56 months, 95% CI, 7.16 to NR) and PD-L1 positive (18.33 months, 95% CI, 6.83 to NR) (p = 0.56). Conclusions: For the first time four commercial kits for ICH analysis were used to test PD-L1 expression in pretreated mRCC pts. Data from these small sample size seem to confirm that PD-L1 in pre-treated mRCC cancer is not a predictive biomarker for selecting pts to receive NIVO-based treatment. Clinical trial information: NCT03469713.

Prognostic Impact of PD-L1 Expression in pN1 NSCLC: A Retrospective Single-Center Analysis.

Simple SummaryThe analysis of prognostic biomarkers (e.g., PD-L1) helps to define treatment for lung cancer patients. To date, these markers have only been examined in metastatic or inoperable situations. We analyzed the PD-L1 expression-levels of tumors from 277 lung cancer patients that underwent curative intent surgery. PD-L1 was identified as a prognostic factor, depending on histologic subtype.The programmed death-ligand 1 (PD-L1) plays a crucial role in immunomodulatory treatment concepts for end-stage non-small cell lung cancer (NSCLC). To date, its prognostic significance in patients with curative surgical treatment but regional nodal metastases, reflecting tumor spread beyond the primary site, is unclear. We evaluated the prognostic impact of PD-L1 expression in a surgical cohort of 277 consecutive patients with pN1 NSCLC on a tissue microarray. Patients with PD-L1 staining (clone SP263) on >1% of tumor cells were defined as PD-L1 positive. Tumor-specific survival (TSS) of the entire cohort was 64% at five years. Low tumor stage (p < 0.0001) and adjuvant therapy (p = 0.036) were identified as independent positive prognostic factors in multivariate analysis for TSS. PD-L1 negative patients had a significantly better survival following adjuvant chemotherapy than PD-L1 positive patients. The benefit of adjuvant therapy diminished in patients with PD-L1 expression in more than 10% of tumor cells. Stratification towards histologic subtype identified PD-L1 as a significant positive predictive factor for TSS after adjuvant therapy in patients with adenocarcinoma, but not squamous cell carcinoma. Routine PD-L1 assessment in curative intent treatment may help to identify patients with a better prognosis. Further research is needed to elucidate the predictive value of PD-L1 in an adjuvant setting.

Pembrolizumab with or without enzalutamide in selected populations of men with previously untreated metastatic castration-resistant prostate cancer harbouring programmed cell death ligand-1 staining: a retrospective study

BackgroundThe purpose of this retrospective study was to evaluate the survival outcomes of pembrolizumab (PEM) plus enzalutamide (ENZ) versus PEM alone in selected populations of men with previously untreated metastatic castration-resistant prostate cancer (mCRPC) harbouring programmed cell death ligand-1 (PD-L1) staining.MethodsConsecutive men with previously untreated mCRPC harbouring PD-L1 staining who underwent treatment with PEM plus ENZ (PE) or PEM alone (PA) at our medical centre from January 1, 2017, to January 31, 2021, were retrospectively identified. Follow-up was conducted monthly during the first year and then every 1 month thereafter. The primary outcomes of the study were overall survival (OS) and progression-free survival (PFS). Secondary outcomes were the frequency of key adverse events (AEs).ResultsIn total, 302 men were retrospectively reviewed, 96 of whom were deemed to be ineligible per the exclusion criteria, leaving 206 men (PE: n = 100, median age 64 years [range, 43–85] and PA: n = 106, 65 years [range, 45–82]) who were eligible for the study. The median follow-up for both groups was 34 months (range, 2–42). At the final follow-up, the median OS was 25.1 months (95% confidence interval [CI], 22.3–27.6) in the PE group versus 18.3 months (95% CI, 16.5–20.9) in the PA group (hazard ratio [HR] 0.56; 95% CI, 0.39–0.80; p = 0.001). A marked distinction was also observed in the median PFS (6.1 months [95% CI, 4.7–7.8] for PE vs. 4.9 months for PA (95% CI, 3.2–6.4) for PA; HR 0.55, 95% CI, 0.41–0.75; p = 0.001). There were noteworthy differences in the rate of the key AEs between the two groups (72.0% for PE vs. 45.3% for PA, p < 0.001). Noteworthy differences were also detected for fatigue events (7.0% in the PE group vs. 0.9% in the PA group, p = 0.025) and musculoskeletal events (9.0% for PE vs. 0.9% for PA, p = 0.007), but these events tended to be manageable.ConclusionsAmong selected populations of men with previously untreated mCRPC harbouring PD-L1 staining, PEM added to ENZ treatment may significantly increase the survival benefits compared with PEM treatment alone regardless of tumor mutation status. The safety profile for PE plus ENZ tends to be manageable.

Impact of Metabolic Activity in Hepatocellular Carcinoma: Association With Immune Status and Vascular Formation.

We evaluated the prognostic value of fluorine‐18 fluorodeoxyglucose (18F‐FDG) positron emission tomography/computed tomography (PET/CT) in hepatocellular carcinoma (HCC). Their association with programmed death ligand 1 (PD‐L1) expression and vascular formation was further investigated. In this retrospective study, using a database of 418 patients who had undergone 18F‐FDG PET/CT before hepatic resection for HCC, immunohistochemical staining of PD‐L1, clusters of differentiation (CD) 8, CD68, and CD34 was performed. Patients with a high maximum standardized uptake value (SUVmax) on 18F‐FDG PET/CT showed a significantly worse recurrence‐free survival (RFS) (hazard ratio [HR]: 1.500; 95% confidence interval [CI]: 1.088‐2.069; P = 0.0133) and overall survival (OS) (HR: 2.259; 95% CI: 1.276‐4.000; P = 0.0052) than patients with a low SUVmax. Logistic regression analysis showed that a high SUVmax in HCC was significantly associated with PD‐L1‐positive expression (odds ratio: 4.407; 95% CI: 2.265‐8.575; P < 0.0001). SUVmax values of HCC were associated with intratumoral CD8‐positive T‐cell counts (P = 0.0044) and CD68‐positive macrophage counts (P = 0.0061). Stratification based on SUVmax, PD‐L1 expression, and the vessels that encapsulate tumor clusters (VETC) status was also significantly associated with RFS and OS. SUVmax, VETC, and PDL1 expression were independently predictive of survival on multivariable analysis. Conclusion: Our large cohort study showed that a high SUVmax on 18F‐FDG PET/CT is associated with a poor clinical outcome and PD‐L1 expression in patients with HCC. Additionally, stratification of patients based on the combination of SUVmax, PD‐L1 expression, and the VETC status predicts poor clinical outcome.

48-Year-Old Woman With Dyspnea and Chest Pain

48-Year-Old Woman With Dyspnea and Chest Pain

Expression of programmed death ligand-1 (PD-L1) in metastatic and postchemotherapy viable testicular germ cell tumors

IntroductionChemotherapy for testicular germ cell tumors (GCT) is highly effective, with few patients who do not respond. Clinical studies to evaluated novel treatments are challenging given the rarity of these patients. Therefore, we sought to evaluate PD-L1 staining on metastatic and postchemotherapy viable testicular GCTs as a surrogate for potential benefit for immunotherapy targeting the PD-1/PD-L1 axis. MethodsEthics research committee approval for this retrospective study was obtained by four participating institutions (CHU de Québec, St. Joseph's Health Care, Halifax Health Science Centre, Johannes Gutenberg University). Patients with viable metastatic testicular GCTs pathology samples were included. Patients with pure teratoma were excluded. PD-L1 staining with the 22C3 clone was evaluated on samples with >100 viable tumor cells using the combined positive score (CPS). ResultsFrom 51 patients identified at participating institutions, 24 postchemotherapy and 18 chemotherapy-naive metastatic samples were available for PD-L1 staining, with 9 matched prechemotherapy samples and 7 matched orchiectomy pathology samples, respectively. The median CPS score was 55.6 (IQR 16–100) for all metastatic samples, 44.9 (IQR 13–100) for postchemotherapy metastatic samples, and 68.8 (IQR 38–100) for chemotherapy-naïve metastatic samples, with the median number of viable tumor cells at 545, 500, and 550, respectively. Differences were not significant between chemotherapy-naïve and postchemotherapy samples (P = 0.07), though among non-seminoma GCT metastatic samples, CPS scores were significantly lower postchemotherapy (P = 0.02). Significant differences among postchemotherapy metastatic tumors were also seen according to predominant subtype, with lower CPS scores for predominant yolk sac and higher values for predominant seminoma and choriocarcinoma. In 7 patients with matched specimens pre- and postchemotherapy, a significant increase in CPS was observed for seminoma (26.7 vs. 81.7, P = 0.045), but not nonseminoma GCTs. Comparing all chemotherapy naïve-samples, PD-L1 expression was higher in metastatic samples versus testicular samples (mean CPS 68.8 vs. 39.8, P = 0.02). This was also seen in matched chemotherapy-naïve samples (mean CPS 77.9 vs. 33.1, P = 0.01). ConclusionOur results suggest that most patients with refractory GCTs postchemotherapy will not benefit from PD-1/PD-L1 immunotherapy. However, the high PD-L1 expression in patients with predominant or pure seminoma post-chemotherapy suggests this may represent a subgroup for whom further trials may be considered.

The importance of histological patterns on PD-L1 staining heterogeneity: Should we use pattern-based approach for selecting tumor samples for PD-L1 testing in lung adenocarcinomas?

Background/aimProgrammed death ligand-1 (PD-L1) is a predictive marker for immunotherapeutic agents. However, heterogeneous staining of PD-L1 can cause false-negative results. The aim of this study is to evaluate the importance of histological patterns on PD-L1 staining heterogeneity in lung adenocarcinomas (LAC). Materials and methods : PD-L1 immunohistochemistry (IHC) stain was performed to two different tissue cores of 128 LAC cases, and cut-off values are given for grouping the cases according to the percentage of staining (1%-10%, 11%-49%, 50%-100%). Staining rates between cores were compared and analyzed by their histological patterns. Also, the relation of the PD-L1 expression with the clinicopathological characteristics of the cases was analyzed. ResultsOverall, PD-L1 expression was observed in 53 of 128 cases (41.4%, 1% cut-off), 23.5% of them were positive at 10% cut-off and 14.1% at 50% cut-off. PD-L1 expression was significantly related to the high grade micropapillary and solid patterns of adenocarcinomas (p:0.01). Staining cut-offs were mostly similar between cores (43/50, 86%) (k:0.843). However, 14% of them were positive only in one core (7 of 50). This false negativity was mostly related to the histological patterns.ConclusionOur data reveal the heterogeneous staining of PD-L1 expression, also micropapillary and solid patterns show higher rates of PDL expression. Therewithal, these findings also highlight the importance of taking into consideration of histological patterns, when choosing a paraffin block for the PDL1.

PD-L1 Testing and Squamous Cell Carcinoma of the Head and Neck: A Multicenter Study on the Diagnostic Reproducibility of Different Protocols.

Simple SummaryThe introduction of therapies with immune checkpoint inhibitors targeting the programmed cell death protein 1 and its ligand (PD-L1) axis in head and neck squamous cell carcinoma prompted the need of reliable bio-selectors to stratify patients that would benefit from these treatments. The assessment of PD-L1 expression through immunohistochemistry represents the most widely used method to perform this task, being recently approved by regulatory authorities. However, borrowing from previous experiences in lung cancer, the heterogeneity of antibodies and platforms used in the routine clinical practice requires a strict multi-institutional harmonization effort. In this setting, the present study is aimed to assess the performances of different PD-L1 staining protocols and the inter-observer reliability for its interpretation.Immune checkpoint inhibitors for blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis are now available for squamous cell carcinoma of the head and neck (HNSCC) in relapsing and/or metastatic settings. In this work, we compared the resulting combined positive score (CPS) of PD-L1 using alternative methods adopted in routine clinical practice and determined the level of diagnostic agreement and inter-observer reliability in this setting. The study applied 5 different protocols on 40 tissue microarrays from HNSCC. The error rate of the individual protocols ranged from a minimum of 7% to a maximum of 21%, the sensitivity from 79% to 96%, and the specificity from 50% to 100%. In the intermediate group (1 ≤ CPS < 20), the majority of errors consisted of an underestimation of PD-L1 expression. In strong expressors, 5 out of 14 samples (36%) were correctly evaluated by all the protocols, but no protocol was able to correctly identify all the “strong expressors”. The overall inter-observer agreement in PD-L1 CPS reached 87%. The inter-observer reliability was moderate, with an ICC of 0.774 (95% CI (0.651; 0.871)). In conclusion, our study showed moderate interobserver reliability among different protocols. In order to improve the performances, adequate specific training to evaluate PD-L1 by CPS in the HNSCC setting should be coordinated.

Programmed Death Ligand 1 Immunohistochemistry in Triple-Negative Breast Cancer: Evaluation of Inter-Pathologist Concordance and Inter-Assay Variability.

PurposeThe programmed death ligand 1 (PD-L1) SP142 assay with a 1% immune cell (IC) cutoff is approved for the selection of advanced triple-negative breast cancer (TNBC) patients for atezolizumab treatment. We aimed to evaluate the interobserver concordance of PD-L1 scoring and inter-assay variability of various PD-L1 assays in TNBC.MethodsThirty patients with primary TNBC were selected, and SP142, SP263, 22C3, and E1L3N assays were performed. PD-L1 staining in ICs and tumor cells (TCs) was scored by 10 pathologists who were blinded to the assay. The interobserver concordance among pathologists and the inter-assay variability of the four PD-L1 assays were analyzed. For SP142, the intraobserver concordance among the six pathologists was analyzed after training.ResultsThe adjusted means of PD-L1 IC scoring ranged from 6.2% to 12.9% for the four assays; the intraclass correlations showed moderate (0.584–0.649) reader concordance. The PD-L1 IC scoring with a 1% cutoff resulted in identical scoring in 40.0%–66.7% of cases and a poor to moderate agreement (Fleiss κ statistic [FKS] = 0.345–0.534) for the four assays. The SP142 assay had the widest range of positive rate (56.5%–100.0%), lowest number of cases with identical scoring, and lowest FKS at 1% cutoff. Pairwise comparison of adjusted means showed significantly decreased PD-L1 staining in SP142 compared with the other assays in both ICs and TCs. As for the intraobserver concordance in the SP142 assay, the overall percent agreement was 87.8% with a 1% IC cutoff. After training, the proportion of cases with identical scoring at a 1% IC cutoff increased to 70.0%; the FKS also increased to 0.610.ConclusionThe concordance of PD-L1 IC scoring among pathologists was low, at the 1% cutoff for the SP142 assay without training. SP142 showed the lowest PD-L1 expression in both IC and TC.

Assessment of PD-L1 expression in patients with neuroblastoma and renal tumors.

Programmed death 1 (PD-1) is a co-receptor which is located at the surface of cells like natural killer, monocytes, T and B cells. It has two ligands including programmed death ligand-1 (PD-L1) and ligand-2 (PD-L2). T cell functions are inhibited by activation of PD-1/PD-L1 pathway and this pathway is used by viruses and some tumor cells in order to escape from immune eradication. In our study we evaluated PD-L1 expression in the tissue specimens of patients with Wilms tumor, neuroblastoma and other renal tumors. Totally 60 patients who were followed up at Gazi University Hospital with the diagnosis of neuroblastoma, Wilms tumor and other renal tumors were included. PD-L1 expression was examined in tumor samples of the patients. Positive staining with PD-L1 was detected only in two male patients. Both of them had neuroblastoma and advanced stage disease. None of the patients with Wilms tumor and other renal tumors had positive PD-L1 staining. Unlike adult tumors, PD-L1 expression is not common in childhood tumors due to differences in immune system between children and adults. Further studies are needed to establish the importance and effects of PD-1/PD-L1 pathway in pediatric tumors.

PD-L1 Expression in Medullary Thyroid Carcinoma and Its Association with Clinicopathological Findings.

< strong > Objective: < /strong > Medullary thyroid carcinoma (MTC) is a rare tumor originating from parafollicular C cells. It has more aggressive biologic behavior than differentiated thyroid carcinomas, and it is insensitive to treatment with radioactive iodine. Vandetanib and cabozantinib are the newly approved tyrosine kinase inhibitors in advanced stages, but novel effective systemic therapeutics could be crucial and needed for the clinical management of these patients. We aimed to evaluate the Programmed death-ligand 1 (PD-L1) expression, which is a novel immunotherapy target, in our MTC cohort, and determine whether it has an association with clinical and pathological features. < strong > Material and Method: < /strong > This retrospective study involved 41 cases of MTC with a median follow-up of 54 months. PD-L1 monoclonal antibody (SP263 clone) was investigated immunohistochemically. Complete and/or partial membranous staining pattern in more than 1% of tumor cells was considered positive. The correlations of PD-L1 expression with clinicopathologic and prognostic features were analyzed. < strong > Results: < /strong > PD-L1 positivity was detected in 5 (12.2%) of 41 tumors. The extent of PD-L1 staining was low ( < 5%) for all tumors. There was no clinicopathologic and prognostic relevance regarding PD-L1 expression in our MTC patients. < strong > Conclusion: < /strong > Although PD-L1 expression could be a potential biomarker to predict the prognosis of various cancers and response to checkpoint inhibitors, we did not find any significant correlation between PD-L1 expression and clinicopathologic features in our cases. Studies with larger patient numbers are still required to perform a more comprehensive analysis.

ARID1A Deficiency Is Associated With High Programmed Death Ligand 1 Expression in Hepatocellular Carcinoma.

The clinicopathological features of carcinomas expressing AT‐rich interaction domain 1a (ARID1A) and programmed death ligand 1 (PD‐L1) in HCC are poorly understood. Here, we examined ARID1A and PD‐L1 expression in surgically resected primary hepatocellular carcinoma (HCC) and the association of ARID1A and PD‐L1 expression with clinicopathological features and patient outcomes. Their association with ARID1A expression and tumor‐associated CD68‐positive macrophage was further explored. Using a database of 255 patients who underwent hepatic resection for HCC, immunohistochemical staining of ARID1A, PD‐L1, and CD68 was performed. We also analyzed the expression PD‐L1 after ARID1A knockdown in HCC cell lines. Samples from 81 patients (31.7%) were negative for ARID1A. Negative ARID1A expression was significantly associated with male sex, high alpha‐fetoprotein, high des‐gamma‐carboxyprothrombin, large tumor size, high rate of poor differentiation, microscopic intrahepatic metastasis, and PD‐L1 expression. In addition, negative ARID1A expression was an independent predictor for recurrence‐free survival, overall survival, and positive PD‐L1 expression. Stratification based on ARID1A and PD‐L1 expression in cancer cells was also significantly associated with unfavorable outcomes. PD‐L1 protein expression levels were increased through phosphoinositide 3‐kinase/AKT signaling after ARID1A knockdown in HCC cells. HCC with ARID1A‐low expression was significantly correlated with high levels of tumor‐associated CD68‐positive macrophage. Conclusion: Our large cohort study showed that ARID1A expression in cancer cells was associated with a poor clinical outcome in patients with HCC, PD‐L1 expression in cancer cells, and tumor microenvironment. Therefore, ARID1A may be a potential molecular biomarker for the selection of patients with HCC for anti‐programmed death 1/PD‐L1 antibody therapy.

Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.

PD-L1 expression in immune cells is a favorable prognostic factor for nasopharyngeal carcinoma.

Programmed death-ligand 1 (PD-L1) has been determined as a reliable prognostic factor for various malignancies. In this study, we aimed to determine the prognostic effect of PD-L1 expression in tumor-infiltrating immune cells (TIICs) of nasopharyngeal carcinoma (NPC) patients. Seventy patients diagnosed with non-metastatic NPC were included in the study. PD-L1 expression on immune cells was analyzed by immunohistochemical method. Patients were categorized into two groups according to the PD-L1 expression level in TIICs (level of PD-L1 staining ≥5% positive vs <5% negative). Median follow-up period was 34 months (range = 1 - 188). 1 and 2 years survival rate were found as 75% and 63% in PD-L1 negative TIICs group (47%), and 85% and 83% in PD-L1 positive TIICs group (53%), respectively. PD-L1 positivity in immune cells (ICs) was detected in 53% of the patients. The survival rate was found better in the PD- L1 positive group compared to the negative group (P = 0.049). In conclusion, the survival rate was found significantly better in the PD-L1 positive TIICs group, compared to the negative group.

CMTM6 Stabilizes PD-L1 Expression and Is a New Prognostic Impact Factor in Hepatocellular Carcinoma.

CKLF‐like MARVEL transmembrane domain containing 6 (CMTM6) was identified as a regulator of programmed death ligand 1 (PD‐L1), which induces antitumor immunity in several cancers. This study aimed to clarify the relationship between CMTM6 and PD‐L1 expression and clinical outcomes in patients with hepatocellular carcinoma (HCC). In total, 259 patients with HCC who had undergone hepatic resection were enrolled. Immunohistochemical staining for CMTM6 and PD‐L1 was performed. The relationships between CMTM6 expression and the clinicopathological characteristics and outcomes were analyzed. Additionally, the stabilization of PD‐L1 expression and regulation of malignant activities by CMTM6 were examined in vitro. Our patients were divided into high (n = 65, 25.1%) and low (n = 194, 74.9%) CMTM6 expression groups. High CMTM6 expression was significantly associated with malignant aggregates, including poor differentiation (P < 0.0001), microscopic intrahepatic metastasis (P = 0.0369), and multiple intrahepatic recurrences (P = 0.0211). CMTM6 expression was significantly correlated with PD‐L1 expression in HCC tissues (P < 0.0001). The patients were classified into three groups: high CMTM6/PD‐L1 positive (n = 21), high CMTM6/ PD‐L1 negative (n = 44), and low CMTM6 (n = 194) expression pattern groups. Overall survival was significantly different among the three groups (P < 0.0001). Additionally, immunohistochemical double staining revealed that CMTM6 and PD‐L1 were co‐expressed on HCC cells. In vitro, PD‐L1 expression was enhanced at late time points in the presence of CMTM6 expression. CMTM6 also regulated epithelial‐to‐mesenchymal transition and stemness phenotypes in HCC cells. Conclusion: Our large cohort study found that CMTM6 co‐expressed with PD‐L1 was strongly associated with the clinical outcome in patients with HCC. The evaluation of CMTM6 combined with PD‐L1 in HCC might be useful for patient selection in immune checkpoint therapy.

An increase of CD8+\u2009T cell infiltration following recurrence is a good prognosticator in HNSCC

Programmed death-ligand 1 (PD-L1) expression and CD8-positive tumor-infiltrating lymphocyte (CD8+ TIL) infiltration are essential biomarkers for immune checkpoint inhibitor therapy. The objective of this study was to compare the expression of those biomarkers between initial and recurrent HNSCCs using paired analysis. Prognostic significance of those immunological changes was also investigated. Forty-two consecutive patients with locally recurrent HNSCCs were included. Immunohistochemical staining of CD8 and PD-L1 was done for both initial and recurrent tumor specimens. The IHC findings were verified with mRNA expression profiling. Also, the prognostic impact was analyzed based on overall survival (OS). Recurrent-to-initial (R/I) ratios of CD8+ TILs and PD-L1 were widely variable. CD8+ TIL density and PD-L1 expression decreased in 59.5% and 69% of patients, respectively (R/I ratio < 1). The R/I ratio of CD8A mRNA was significantly higher in patients with a CD8 R/I ratio > 1 (1.7 ± 1.5 vs. 0.6 ± 0.6, p = 0.042). CD8 R/I ratio (> 1) was a good prognosticator for OS (HR 0.293, 95% CI 0.091–0.945, p = 0.040). CD8+ TIL infiltration and PD-L1 expression changed variably following local recurrence of HNSCC. The increase of CD8+ TILs at recurrence was an excellent independent prognosticator.

Effects of immunohistochemical conditions on the results of PD-L1 (22C3) staining

Objective: To investigate the optimal experimental conditions (including antigen retrieval time, antibody titers and antibody incubation time) for reliable detection of programmed death-ligand 1 (PD-L1) expression using PD-L1 (22C3) antibody concentrate, and to establish a laboratory developed test for PD-L1 detection. Methods: Using Dako PD-L1 IHC 22C3 pharmDX staining procedure and scoring guidelines as the standard reference (group A), the PD-L1 expression in 25 tissue specimens (including 15 lung cancer tissues, 5 tonsil tissues and 5 placenta tissues) was detected with Flex+/HRP detection kit (EnVision) under 8 different experimental conditions (groups B1 to B8). The staining results were then compared to those in group A. Results: In group B1, 3 tissue samples showed the percentages of PD-L1 positive tumor cells were similar to those in group A, while the percentages of PD-L1 positive tumor cells were lower than those in group A in the other samples. In group B7, two case showed a positive rate higher than that in group A that was also above the positive cut-off value, and the rest of the samples had a percentage of PD-L1 positive tumor cells slightly higher than that in group A, but still below the positive cut-off value. The staining results of group B8 were the closest to those of group A compared with the other groups. Although the percentages of PD-L1 positive tumor cells in the B2 to B6 groups were decreased in various degrees as compared with group A, they were still concordant with group A's classification (positive vs. negative) and would not change the choice of clinical treatments. Conclusions: The experimental conditions are associated with detection rate of PD-L1 expression using 22C3 antibody. In the present study, the most-suitable alterative conditions in the PD-L1 detection using 22C3 antibody concentrate are those applied in the group B8 (including antigen retrieval in Dako PT Link tank at 97 ℃, pH 6.0 for 40 min and incubation with 22C3 antibodies (1∶100 dilution) at room temperature for 60 min, incubation with EnVision Flex+Linker at room temperature for 30 min, incubation with EnVision/HRP at room temperature for 30 min and DAB staining for 5 min), which could provide reliable results at minimum costs.

Clinical significance of PD-L1 expression in patients with surgically resected non-small cell lung cancer.

To explore the expression of programmed death ligand-1 (PD-L1) as well as the correlation between the expression and the clinicopathological features or prognosis in non-small cell lung cancer (NSCLC). The expression of PD-L1 protein in 254 cases of surgically resected lung adenocarcinoma (L-ADC), 228 cases of surgically resected lung squamous cell cancer (L-SCC), and 99 cases of non-cancerous control lung tissues was detected with immunohistochemical SP method. The correlation between the PD-L1 expression and clinicopathological features was analyzed. Kaplan-Meier univariate and Cox multivariate regression analyses were performed to assess the prognosis of patients with L-ADC and L-SCC, respectively. Positive percentage of PD-L1 protein expression was higher in the tissues of L-ADC and L-SCC than that in the non-cancerous control lung tissues respectively (both P<0.05). Positive percentage of PD-L1 protein expression was higher in the tissues of L-SCC than that in the tissues of L-ADC (P<0.05). However, there were no correlation between the expression of PD-L1 and age, gender, pathological grades, clinical stages, and lymph node metastasis in L-ADC and L-SCC respectively (all P>0.05). Overall survival rate was evidently lower in the L-ADC patients with positive expression of PD-L1 protein compared to the patients with negative staining of PD-L1 (P<0.01). Multivariate Cox regression analysis further identified that the L-ADC patients of positive expression of PD-L1 protein had a poor prognosis (P<0.01). The positive percentage of PD-L1 protein expression is higher in the L-SCC patients than that in the L-ADC patients. Positive expression of PD-L1 protein can be served as an independent prognostic factor of poor prognosis in the patients with L-ADC.

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.