Clinical significance of inter-assay discrepancy in PD-L1 evaluation for the efficacy of pembrolizumab in advanced NSCLC with high PD-L1 expression.

INTRODUCTION: Programmed cell death ligand-1 (PD-L1) expression is a predictive biomarker for the efficacy of anti-programmed cell death receptor-1/PD-L1 antibodies in advanced non-small cell lung cancer (NSCLC). Although several assays have been approved for evaluating PD-L1 expression status, inter-assay discordance has been observed between some assays. The clinical significance of these discrepancies is still unclear. METHODS: We retrospectively reviewed treatment-naïve NSCLC patients whose PD-L1 expression was evaluated using both 22C3 and SP142 assays. Among those, efficacy analysis was performed for patients with PD-L1 tumor proportion score (TPS) ≥ 50 % (22C3), who had received first-line pembrolizumab monotherapy. Additionally, transcriptome analysis was conducted in the available tumors with TPS ≥ 50 % to investigate the distinct immune profiles that accompany inter-assay discordance. RESULTS: In total, 611 patients were eligible. Among 198 patients with TPS ≥ 50 %, 91 (46 %) had tumor cell score ≤ 1 (SP142, i.e., inter-assay discrepancy). In the 52 patients who received first-line pembrolizumab monotherapy, treatment efficacy was significantly lower in patients with the discrepancy than that in those without (objective response rate: 18 % vs. 83 %, p < 0.001; median progression-free survival [months]: 3.2 vs. 8.3, p < 0.001). Transcriptome analysis revealed significantly more CD274 splice variants with aberrant 3'-terminal sequences in tumors with the inter-assay discrepancy than in those without. CONCLUSION: The inter-assay discrepancy in the PD-L1 status of tumor cells between the 22C3 and SP142 assays, reflecting an imbalance in the CD274 splice variants, could be a biomarker for primary resistance against pembrolizumab monotherapy in high PD-L1-expressing NSCLCs.

A Systemic Immune Inflammation Index and PD-L1 (SP142) Expression as a Potential Combined Biomarker of the Clinical Benefit of Chemo-Immunotherapy in Extensive-Stage Small-Cell Lung Cancer.

Background: No studies have identified combined biomarkers that may be more reasonable for the assessment of current chemo-immunotherapy in patients with extensive stage small-cell lung cancer (ES-SCLC). Methods: This study was conducted to investigate a combined biomarker with prognostic or predictive value in ES-SCLC. We determined the best independent prognostic biomarker among the four complete blood-count-derived inflammatory biomarkers (CBC-IBs). Subsequently, we analyzed the prognostic or predictive value of combining this independent CBC-IB with PD-L1 (SP142) expression. We prospectively assessed the SP142 analyses in tumor samples at diagnosis. Results: All in all, 55 patients with ES-SCLC were classified into four groups according to the systemic immune inflammation index (SII) (low/high) and SP142 (positive/negative). The best survival was observed in the low-SII/ SP142-positive group, whereas the worst survival was observed in the high-SII/SP142-negative group (p = 0.002). The combined SII-SP142 biomarker was better for predicting both survival and disease progression in patients with ES-SCLC. Conclusions: The combined SII-SP142 biomarker can be readily and universally obtained at a low cost in clinical practice, without requiring advanced genomics technology or specialized expertise. Although further studies are needed to confirm that the combined SII-SP142 biomarker is widely applicable, it should help clinicians to identify the best patients for combined chemotherapy with atezolizumab in ES-SCLC.

PD-L1 immunohistochemical expression in bladder urothelial cancer with SP263, SP142 and 22C3 antibodies: A comparative study.

Programmed death ligand 1 (PD-L1) is currently the only biomarker used for the selection of patients with bladder urothelial cancer for immunotherapy. Several platforms, antibodies and scores are currently available for the evaluation of the expression of PD-L1 in immunohistochemistry (IHC). In this study three different antibodies (SP263, SP142 and 22C3) were compared to establish their performances and concordance rates. Twenty-four consecutive cases of surgically resected urothelial cancers of the bladder were enrolled. All cases were revised, and appropriate tumor areas were selected for IHC. Three commercially available PD-L1 antibodies were tested: 22C3 pharmDx with Dako Autostainer Link 48 (Dako, Carpinteria, Ca), and SP263 and SP142 with the Ventana BenchMark (Ventana Medical Systems, Tucson, AZ) platform. All slides were evaluated by an expert pathologist and both the tumor proportion score (TPS) and the combined positive score (CPS) were determined and compared at two different cut-off levels (≥ 1 and ≥ 10). The SP263 and 22C3 clones produced more positive results with the CPS and TPS scores, respectively. The CPS score identified more positive cases than the TPS score, irrespectively of the clone or the cut-off used; the difference was statistically significant in both the SP263 and SP142 clones with the ≥1 cut-off. No statistically significant differences were found between the clones when the ≥1 cut-off was used, irrespectively of the score. At the contrary, a statistically significant difference (p = 0.024) and a trend to significance (p = 0.082) were respectively found for the TPS and CPS scores, when the SP22C3 and the SP142 clones were compared at a cut-off level of ≥10. The ICC test using CPS was 0.676 and 0.578 for the ≥1 and ≥ 10 cut-offs respectively, and 0.729 and 0.467 respectively for the same cut-offs using TPS. This suggests that the three antibodies under investigation cannot be used interchangeably, especially the 22C3 and SP142 clones which showed statistically significant difference when TPS was tested at a ≥ 10 cut-off.

PD-L1 (SP142) Expression in Primary and Recurrent/Metastatic Triple-Negative Breast Cancers and Its Clinicopathological Significance.

PURPOSE: The programmed death-ligand 1 (PD-L1) SP142 assay identifies patients with triple-negative breast cancer (TNBC) who are most likely to respond to the anti-PD-L1 agent atezolizumab. We aimed to compare PD-L1 (SP142) expression between primary and recurrent/metastatic TNBCs and elucidate the clinicopathological features associated with its expression. MATERIALS AND METHODS: Primary and recurrent/metastatic TNBCs tested with PD-L1 (SP142) were collected, and clinicopathological information of these cases was obtained through a review of slides and medical records. RESULTS: PD-L1 (SP142) positivity was observed in 50.9% (144/283) of primary tumors and 37.8% (31/82) of recurrent/metastatic TNBCs with a significant difference. Recurrent or metastatic sites were associated with PD-L1 positivity, with high PD-L1 positivity in the lung, breast, and soft tissues, and low positivity in the bone, skin, liver, and brain. When comparing PD-L1 expression between primary and matched recurrent/metastatic TNBCs using 55 paired samples, 20 cases (36.4%) showed discordance; 10 cases revealed positive conversion, and another 10 cases revealed negative conversion during metastatic progression. In primary TNBCs, PD-L1 expression was associated with a higher histologic grade, lower T category, pushing border, and higher tumor-infiltrating lymphocyte infiltration. In survival analyses, PD-L1 positivity, especially high positivity, was found to be associated with favorable prognosis of patients. CONCLUSION: PD-L1 (SP142) expression was lower in recurrent/metastatic TNBCs, and substantial cases showed discordance in its expression between primary and recurrent/metastatic sites, suggesting that multiple sites may need to be tested for PD-L1 (SP142) when considering atezolizumab therapy. PD-L1 (SP142)-positive TNBCs seems to be associated with favorable clinical outcomes.

Comparison of SP142 and 22C3 PD-L1 assays in a population-based cohort of triple-negative breast cancer patients in the context of their clinically established scoring algorithms.

BACKGROUND: Immunohistochemical (IHC) PD-L1 expression is commonly employed as predictive biomarker for checkpoint inhibitors in triple-negative breast cancer (TNBC). However, IHC evaluation methods are non-uniform and further studies are needed to optimize clinical utility. METHODS: We compared the concordance, prognostic value and gene expression between PD-L1 IHC expression by SP142 immune cell (IC) score and 22C3 combined positive score (CPS; companion IHC diagnostic assays for atezolizumab and pembrolizumab, respectively) in a population-based cohort of 232 early-stage TNBC patients. RESULTS: The expression rates of PD-L1 for SP142 IC ≥ 1%, 22C3 CPS ≥ 10, 22C3 CPS ≥ 1 and 22C3 IC ≥ 1% were 50.9%, 27.2%, 53.9% and 41.8%, respectively. The analytical concordance (kappa values) between SP142 IC+ and these three different 22C3 scorings were 73.7% (0.48, weak agreement), 81.5% (0.63) and 86.6% (0.73), respectively. The SP142 assay was better at identifying 22C3 positive tumors than the 22C3 assay was at detecting SP142 positive tumors. PD-L1 (CD274) gene expression (mRNA) showed a strong positive association with all two-categorical IHC scorings of the PD-L1 expression, irrespective of antibody and cut-off (Spearman Rho ranged from 0.59 to 0.62; all p-values < 0.001). PD-L1 IHC positivity and abundance of tumor infiltrating lymphocytes were of positive prognostic value in univariable regression analyses in patients treated with (neo)adjuvant chemotherapy, where it was strongest for 22C3 CPS ≥ 10 and distant relapse-free interval (HR = 0.18, p = 0.019). However, PD-L1 status was not independently prognostic when adjusting for abundance of tumor infiltrating lymphocytes in multivariable analyses. CONCLUSION: Our findings support that the SP142 and 22C3 IHC assays, with their respective clinically applied scoring algorithms, are not analytically equivalent where they identify partially non-overlapping subpopulations of TNBC patients and cannot be substituted with one another regarding PD-L1 detection. Trial registration The Swedish Cancerome Analysis Network - Breast (SCAN-B) study, retrospectively registered 2nd Dec 2014 at ClinicalTrials.gov; ID NCT02306096.

Discordance in PD-L1 expression using 22C3 and SP142 assays between primary and metastatic triple-negative breast cancer.

AIMS: SP142 and 22C3 assays are approved companion diagnostic assays for anti-PD-1/PD-L1 therapy selection in metastatic triple-negative breast cancer (TNBC). The discordance in PD-L1 status between primary and metastatic tumors in the same patient has been poorly characterized. Here, we examined the concordance of PD-L1 status between the two assays and between primary tumors and metastases for each assay. METHODS: We retrospectively evaluated tumor samples from 160 patients with TNBC, including 45 patients with paired primary and metastatic tumors. PD-L1 status was assessed using SP142 and 22C3 assays, to determine the immune cell (IC) score, tumor cell (TC) score (SP142 and 22C3), and combined proportion score (CPS: 22C3). RESULTS: The concordance of PD-L1 positivity at diagnostic cutoffs for SP142 (IC ≥ 1) and 22C3 (CPS ≥ 10) was substantial (κ = 0.80) in primary tumors and moderate (κ = 0.60) in metastatic tumors. In comparison, between primary and metastatic tumors, the concordance with 22C3 was moderate (κ = 0.50), whereas that with SP142 was poor (κ = -0.03). Among patients who were PD-L1 negative for both assays in primary tumors, 7/30 (23.3%) were PD-L1 positive for both or either 22C3 or SP142 in the metastatic tumors. CONCLUSIONS: The inter-assay concordance of PD-L1 positivity at diagnostic cutoffs was substantial in primary tumors and moderate in metastatic tumors. Discordance between PD-L1 status in primary and metastatic tumors was frequently observed, especially with SP142. Some patients with a PD-L1-negative status in primary tumors may still be candidates for immunotherapy, depending on the PD-L1 status in their metastatic tumors.

[Prognostic significance of PD-L1 expression as a potential predictor of survival in gastric cancer]. / Prognosticheskaya znachimost' ekspressii PD-L1 kak potentsial'nogo prediktora vyzhivaemosti pri rake zheludka.

BACKGROUND: Currently, PD-L1 expression in patients with tumors of various localizations is being actively studied. Studies on the expression of PD-L1 detected by clones SP142 and SP263 in gastric cancer (for the drugs atezolizumab and durvalumab, respectively) are rare in the literature. The prognostic role of PD-L1 expression in patients who were not treated with immune checkpoint inhibitors has also not been investigated. OBJECTIVE: To determine the expression level of PD-L1 (clones SP263 and SP142, Roche Ventana) in gastric cancer specimens and evaluate its effect on overall survival in patients who did not receive adjuvant therapy with immune checkpoint inhibitors. MATERIAL AND METHODS: The study included 131 patients with a verified diagnosis of gastric cancer. The material obtained from 127 patients was stained with antibodies to PD-L1 SP263, and from 126 patients - with antibodies to PD-L1 SP142. A multivariate Cox regression model with Wald's step-by-step exclusion algorithm was used to evaluate predictors of survival. RESULTS: The total five-year survival rate of patients in the PD-L1-negative tumor group was significantly lower than the total five-year survival rate of patients in the PD-L1-positive tumor group, which was 50.0% and 40.0% also for both clones (p=0.027). An increase in the expression of PD-L1 clone SP263, determined by both the CPS and TPS method, reduces the chances of death by 1.35 times (p=0.02) and 1.61 times (p=0.004), respectively. An increase in the expression of PD-L1 clone SP142, determined by the CPS method, reduces the chances of death by 1.54 times (p=0.005). CONCLUSION: The survival rate of patients in the group of PD-L1-positive tumors is significantly higher than in patients in the group of PD-L1-negative tumors. Elevated PD-L1 expression, as assessed by the SP263 and SP142 clones, is an important prognostic marker that predicts a higher chance of overall survival for patients, even though these patients are not receiving immune checkpoint inhibitors adjuvant therapy.

Tumor-Infiltrating Lymphocytes and PD-L1 Expression in Pleomorphic Lobular Breast Carcinoma.

BACKGROUND: The prognostic and predictive role of stromal tumor-infiltrating lymphocytes (sTILs) is undetermined in pleomorphic invasive lobular cancer (pILC). The same applies for the expression of PD-1/PD-L1 in this rare breast cancer subtype. Here, we aimed to investigate the expression of sTILs and analyze the PD-L1 expression levels in pILC. METHODS: Archival tissues from sixty-six patients with pILC were collected. The sTIL density was scored as a percentage of tumor area using the following cut-offs: 0%; <5%; 5-9%; and 10-50%. The PD-L1 expression was analyzed using IHC on formalin-fixed, paraffin-embedded tissue sections using SP142 and 22C3 antibodies. RESULTS: A total of 82% of the sixty-six patients were hormone receptor positive and 8% of cases were triple negative (TN), while 10% showed human epidermal growth factor receptor 2 (HER2) amplification. sTILs (≥1%) were present in 64% of the study population. Using the SP142 antibody, 36% of tumors demonstrated a positive PD-L1 score of ≥1%, and using the 22C3 antibody, 28% had a positive PD-L1 score of ≥1. There was no correlation between sTILs or PD-L1 expression and tumor size, tumor grade, nodal status, expression of estrogen receptor (ER), or amplification of HER2. Our data did not show any difference in survival between the three molecular subtypes of pILC with respect to sTILs and PD-L1 expression. CONCLUSION: This study shows that pILCs show some degree of sTILs and PD-L1 expression; however, this was not associated with a survival improvement. Additional large trials are needed to understand immune infiltration in lobular cancer, especially in the pleomorphic subtype.

Enhanced PD-L1 expression on tumor cells in primary CD30-positive cutaneous large T-cell lymphoma: a report of lymph node lesions of four cases.

Scarce data are available regarding neoplastic PD-L1 (nPD-L1, clone SP142) expression in cutaneous T-cell lymphoma. We recently documented a possible association of increased nPD-L1 expression with tumor progression to secondary nodal involvement in two cases of CD30-positive primary cutaneous large T-cell lymphoma (PC-LTCL) (Pathol Int 2020;70:804). Notably, the nodal sites exhibited classic Hodgkin lymphoma (CHL) mimicry related to both morphology and tumor microenvironment (TME), i.e., abundant PD-L1-positive tumor-associated macrophages and low-level PD-1 expression on T-cells. Immunohistochemistry highlighted distinctly different nPD-L1 positivity between the cutaneous and nodal lesions. In the present study, we aimed to validate this unique phenomenon in a larger series of four cases with FISH and targeted-capture sequencing (targeted-seq) analysis. We retrospectively identified two more cases of CD30-positive PC-LTCL with secondary nodal involvement among all patients consecutively diagnosed between 2001-2021. All cases immunohistochemically exhibited elevated nPD-L1 expression on ≥50% of lymphoma cells in nodal tumors, clearly contrasting with the scarce nPD-L1 positivity (≤1%) in cutaneous tumors. Moreover, all nodal lesions exhibited CHL-like TME, with abundant PD-L1-positive tumor-associated macrophages and low-level PD-1 expression on T cells, although the CHL-like morphology was limited in the two original cases. None showed CD274/PD-L1 copy number alteration by FISH analysis, or structural variations of PD-L1 3'-UTR by targeted-seq analysis. These findings indicated that nPD-L1 expression is linked with tumor progression and CHL-like TME in nodal involvement of PC-LTCL. Interestingly, one autopsied case exhibited heterogeneity of nPD-L1 expression at different disease sites.

Inter- and Intra-Observer Agreement of PD-L1 SP142 Scoring in Breast Carcinoma-A Large Multi-Institutional International Study.

The assessment of PD-L1 expression in TNBC is a prerequisite for selecting patients for immunotherapy. The accurate assessment of PD-L1 is pivotal, but the data suggest poor reproducibility. A total of 100 core biopsies were stained using the VENTANA Roche SP142 assay, scanned and scored by 12 pathologists. Absolute agreement, consensus scoring, Cohen's Kappa and intraclass correlation coefficient (ICC) were assessed. A second scoring round after a washout period to assess intra-observer agreement was carried out. Absolute agreement occurred in 52% and 60% of cases in the first and second round, respectively. Overall agreement was substantial (Kappa 0.654-0.655) and higher for expert pathologists, particularly on scoring TNBC (6.00 vs. 0.568 in the second round). The intra-observer agreement was substantial to almost perfect (Kappa: 0.667-0.956), regardless of PD-L1 scoring experience. The expert scorers were more concordant in evaluating staining percentage compared with the non-experienced scorers (R2 = 0.920 vs. 0.890). Discordance predominantly occurred in low-expressing cases around the 1% value. Some technical reasons contributed to the discordance. The study shows reassuringly strong inter- and intra-observer concordance among pathologists in PD-L1 scoring. A proportion of low-expressors remain challenging to assess, and these would benefit from addressing the technical issues, testing a different sample and/or referring for expert opinions.

Expression of Three Clones of PD-L1 in Lung Cancer: A Single-center Experience.

BACKGROUND/AIM: Programmed cell death ligand 1 (PD-L1) is an immune checkpoint protein involved in immune evasion of malignant tumors. Confirmation of PD-L1 expression in non-small cell lung cancer (NSCLC) is necessary for the determination of immunotherapy using immune checkpoint inhibitors (ICIs). PDL-1 expression is currently analyzed by immunohistochemistry and is the only available biomarker that can guide the treatment of NSCLC using ICIs. The present study was conducted to compare the expression of three different commercial clones of PD-L1 in order for immunohistochemistry (IHC) for these clones to become more reliable for surgical pathologists. MATERIALS AND METHODS: This study examined the expression of PD-L1 in 76 cases of resected lung cancer using IHC. Three clones were examined: SP263, SP142, and 22C3PharmDx, which are commercially approved for quantifying PD-L1 expression in lung cancer. RESULTS: Of the 76 patients whose samples were evaluated for PD-L1 using the IHC 22C3pharmDx assay, 19 (25.0%) had a tumor proportion score (TPS) of ≥50% and 41 (53.9%) had a PD-L1 TPS of ≥1%. Furthermore, using the SP263, 48.7% had a TPS of ≥1% and 18.4% of >50%. The SP142 assay was used to evaluate tumor cells (TCs) and immune cells (ICs). Twenty (26.3%) cases were positive for TCs and 25 (32.9%) were reactive for ICs. CONCLUSION: These three commercial PD-L1 clones are comparable for detecting primary targets for anti-tumor immunotherapies. Careful evaluation by a pathologist is necessary to minimize misinterpretation errors.

PD-L1 expression in muscle invasive urothelial carcinoma: Comparison of SP142 and SP263 assay.

Introduction: High-grade urothelial carcinoma has a different molecular pathway than superficial low grade urothelial carcinoma, and is characterized by genomic instability. The high tumor mutation burden leads to neoantigen formation, evoking an immune response. The immune response has been keenly studied in last two decades and programmed death ligand-1 (PDL-1) has emerged as acceptable immunohistochemical marker for assessment of response to therapy, prognostication and patient selection for immunotherapy. The targeting of PD-1 and PDL-1 by checkpoint inhibitors (CPIs) is an attractive strategy to unblock the inhibitor and induce cytotoxic cell death. However, the presence of complementary and companion diagnostic testing with multiple PDL-1 assays and platforms for various CPIs make a diagnostic quagmire. Thus, it is the need of hour to harmonize these assays. In this undertaken study we evaluated the concordance in PD-L1 expression between the two PD-L1 clones: SP263 and SP142, in treatment naïve muscle invasive bladder cancer (MIBC). Methods: We evaluated Ventana PD-L1 "SP263 and SP142" qualitative immunohistochemical assay using rabbit monoclonal anti-PD-L1 clones in evaluation of PDL-1 immunoexpression on Ventana autostainer platform. The study includes 30 muscle invasive urothelial carcinomas, with 10 of 30 having nodal metastasis. Results: SP263 assay was statistically more sensitive than SP142 for tumor cell (TC) scoring (P = 0.0009), whereas SP142 was more sensitive for immune cell (IC) scoring (P = 0.0067). There was no statistical significant discordance for TC or IC scoring between primary tumor and metastatic lymph node. Conclusion: PD-L1 testing status can be done on both primary tumor and metastatic site, however in metachronous metastatic setting, testing on recent metastatic site should be preferred. The harmonization of immunoexpression between 2 PD-L1 clones could not be achieved.

Clinicopathological and Prognostic Significance of Programmed Death Ligand-1 SP142 Expression in 132 Patients With Triple-negative Breast Cancer.

BACKGROUND/AIM: The prognostic value of programmed death ligand-1 (PD-L1) expression in triple-negative breast cancer (TNBC) has not been sufficiently investigated. In this study, we examined whether PD-L1 expression status is associated with clinicopathological features and outcomes of patients with TNBC. PATIENTS AND METHODS: Immunostaining for PD-L1 SP142 was performed on tissue microarrays containing 132 TNBC samples. High PD-L1 expression was defined as ≥10% of the tumor area occupied by PD-L1-expressing cells. RESULTS: Thirty-five (26.5%) patients showed high PD-L1 SP142 expression on immune cells (ICs). High IC PD-L1 expression was significantly correlated with smaller tumor size (p=0.030), absence of lymphovascular invasion (p=0.024), and fewer lymph node metastases (p=0.002). Multivariate survival analysis revealed that high IC PD-L1 expression independently predicted better disease-free survival (DFS) of TNBC patients. CONCLUSION: High PD-L1 SP142 expression on ICs was significantly associated with favorable clinicopathological parameters and better outcomes in patients with TNBC. Our observations suggest that high IC PD-L1 expression can be used as an independent prognostic marker for predicting better DFS in patients with TNBC.

Concordance of PD-L1 expression in triple-negative breast cancers in Chinese patients: A retrospective and pathologist-based study.

OBJECTIVE: To compare the expression of programmed cell death ligand 1 (PD-L1) in different paraffin blocks from the same triple-negative breast cancers (TNBC) specimen and between matched primary tumors and lymph node metastases (LNMets). We also aim to determine the interobserver agreement between pathologists trained on PD-L1 (SP142) assay in assessing TNBC. METHODS: 426 histologically confirmed TNBC cases, in which 85 have LNMets, were included in this study. A PD-L1 (SP142) assay was used to identify PD-L1 expression on tumor infiltrating immune cells (IC) and also on tumor cells (TC) in primary tumors and LNMets of TNBC by two trained pathologists. PD-L1 scoring and assessment were based on criteria in IMpassion 130 trial criteria. Concordance of PD-L1 expression in TNBC were analyzed using Kappa-test and assessed by the Kappa value. RESULTS: Prevalence of positive PD-L1 expression (PD-L1 +) on tumor-infiltrating immune cells (PD-L1 IC+) (IC≥1%) in LNMets (49.4%) was higher than in the matched primary tumors (38.9%). Concordance of PD-L1 expression on IC between the two paraffin blocks from the same primary tumor specimen was substantial (P < 0.000, Kappa = 0.627) and was identified in 83.1% (108/130) of the selected cases. For TNBC cases with matched primary and LNMets blocks, the concordance of PD-L1IC scoring between the two blocks was moderate (P < 0.000, Kappa = 0.434). Interobserver agreement of PD-L1 assessment was 78.2% (P < 0.000, Kappa = 0.567) in primary tumors and 61.4% (P < 0.000, Kappa = 0.253) in the matched LNets. CONCLUSION: Substantial intratumor concordance of PD-L1 scoring of the primary tumors in TNBC patients was determined, implying that immunohistochemically detection using one representative block of the primary tumor should be enough to assign the expression status of PD-L1 in clinical practice. The prevalence of PD-L1 + in lymph node metastases (LNMets) was higher than in the matched primary tumors, implying that PD-L1 detection in LNMets may provide additional PD-L1 expression information, especially in TNBC cases with PD-L1- in the matched primary breast tumors. Interobserver agreement of PD-L1 scoring in primary tumors was moderate while only fair in LNMets, implying that the additional training for PD-L1 assessment of TNBC LNMets specimens is recommended to enhance interobserver agreement. DATA AVAILABILITY: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

SP142 PD-L1 Assays in Multiple Samples from the Same Patients with Early or Advanced Triple-Negative Breast Cancer.

PURPOSE: The discernible PD-L1 staining of tumor-infiltrating lymphocytes occupying ≥ 1% of the tumor area is considered SP142 PD-L1 positive for atezolizumab, and the PD-L1 status of multiple samples within a single patient could be discrepant. In this study, we evaluated the PD-L1 status by using the SP142 clone in serially collected matched samples from the same individuals with early or metastatic triple-negative breast cancer (TNBC). METHOD: the SP142 PD-L1 assay was performed using biopsies and surgical specimens from 77 patients with early TNBC. Among these patients, 47 underwent upfront surgery, and 30 underwent neoadjuvant chemotherapy (NAC) between biopsy and surgery. PD-L1 assays were performed at least twice in 8/12 (66.7%) patients with metastatic TNBC treated with atezolizumab and nab-paclitaxel. RESULTS: Of the 47 patients who underwent upfront surgery, 15/47 (31.9%) had PD-L1+ on biopsied samples. PD-L1+ rates in the biopsy and surgical specimens increased to 66.0% (33 of 47) after subsequent surgery. Similarly, in the 30 patients with residual invasive cancer who underwent neoadjuvant chemotherapy, the PD-L1+ rate increased from 46.6% at baseline to 74.2% after surgery. In the 77 patients with early TNBC, multiple PD-L1 testing in the biopsies and surgical specimens significantly increased the number of patients with PD-L1+ compared with the number of patients with PD-L1+ assessed with initial biopsy samples alone (68.8% vs. 37.6%; p = 0.00002). Among the metastatic TNBC patients, those with constant PD-L1+ over 1% positivity in multiple samples showed a response which was longer than 12 months. CONCLUSIONS: Our findings reveal the heterogeneous SP142 PD-L1 expression in TNBC and suggest that PD-L1 evaluation in baseline biopsy might be insufficient to represent the PD-L1 status of whole tumors. In TNBC, vigorous PD-L1 examination using multiple available tumor samples could identify more patients eligible for immune checkpoint blockade.

PD-L1 (SP142 and 22C3) immunohistochemistry in clinical metastatic triple-negative or low hormone receptor breast carcinomas: experience from a large academic institution.

The discovery of new immune checkpoint molecules has led to the emergence of new treatments for metastatic breast carcinoma. We aimed to investigate PD-L1 (SP142 and 22C3) expression in 77 clinical metastatic triple-negative or low hormone receptor (<10%) breast carcinomas (TNBC/LHRBC). SP142 was positive in 23.4% of cases (18/77). SP142-positive (SP142+) cases showed lower liver metastasis and androgen receptor expression, but increased tumor-infiltrating lymphocytes than SP142-negative (SP142-) cases in univariate analysis, but only increased tumor-infiltrating lymphocytes in multivariate analysis. 22C3 testing was available in 21 cases including 14 with combined positive score (CPS) ≥10 (9 SP142- and 5 SP142+) and 7 with CPS <10 (7 SP142-). Ten (13%) patients received immunotherapy including 8 SP142+ (7 with atezolizumab and 1 with pembrolizumab) and 2 SP142- cases (2 with pembrolizumab). Survival data showed a trend of increased survival rate in SP142+ (72.2%) when compared to SP142- patients (55.9%). Our study provides unique insights into the distribution of PD-L1 staining in metastatic breast carcinomas in a real-world clinical setting.

Immunohistochemical assessment of PD-L1 expression using three different monoclonal antibodies in triple negative breast cancer patients.

BACKGROUND: PD-L1 receptor expression in breast cancer tissue can be assessed with different anti-human PD-L1 monoclonal antibodies. The performance of three specific monoclonal antibodies in a head-to-head comparison is unknown. In addition, a potential correlation of PD-L1 expression and clinico-pathological parameters has not been investigated. METHODS: This was a retrospective study on tissue samples of patients with histologically confirmed triple negative breast cancer (TNBC). PD-L1 receptors were immune histochemically stained with three anti-human PD-L1 monoclonal antibodies: 22C3 and 28-8 for staining of tumor cell membranes (TC) and cytoplasm (Cyt), SP142 for immune cell staining (IC). Three different tissue samples of each patient were evaluated separately by two observers in a blinded fashion. The percentage of PD-L1 positive tumor cells in relation to the total number of tumor cells was determined. For antibodies 22C3 and 28-8 PD-L1 staining of 0 to < 1% of tumor cells was rated "negative", 1-50% was rated "positive" and > 50% was rated "strong positive". Cyt staining was defined as "negative" when no signal was observed and as "positive", when any positive signal was observed. For IC staining with SP142 all samples with PD-L1 expression ≥ 1% were rated as "positive". Finally, the relationship between PD-L1 expression and clinico-pathological parameters was analyzed. RESULTS: Tissue samples from 59 of 60 enrolled patients could be analyzed. Mean age was 55 years. Both the monoclonal antibodies 22C3 and 28-8 had similar properties, and were positive for both TC in 13 patients (22%) and for Cyt staining in 24 patients (40.7%). IC staining with antibody SP142 was positive in 24 patients (40.7%), who were also positive for Cyt staining. The differences between TC and Cyt staining and TC and IC staining were significant (p = 0.001). Cases with positive TC staining showed higher Ki67 expression compared to those with negative staining, 40 vs 30%, respectively (p = 0.05). None of the other clinico-pathological parameters showed any correlation with PDL1 expression. CONCLUSIONS: Antibodies 22C3 and 28-8 can be used interchangeably for PD-L1 determination in tumor cells of TNBC patients. Results for Cyt staining with 22C3 or 28-8 and IC staining with SP142 were identical. In our study PD-L1 expression correlates with Ki67 expression but not with OS or DFS.

[Expression comparison and clinical significance of PD-L1 (22C3) and PD-L1 (SP142) in triple negative breast cancer].

Objective: To investigate the expression of programmed death ligand-1 (PD-L1, SP142) and PD-L1 (22C3) in triple-negative breast cancer (TNBC), and analyze their correlation with the clinicopathological factors and prognosis. Methods: The clinicopathologic data of 259 patients with TNBC treated in Cancer Hospital from August 2010 to December 2013 were collected. Whole section of surgical tissue samples were collected to conduct PD-L1 (SP142) and PD-L1 (22C3) immunohistochemical (IHC) staining. The PD-L1 expression in tumor cells and tumor infiltrating immune cells were visually assessed respectively, the relationship between PD-L1 expression and clinicopathologic characterizes were analyzed. Univariable and multivariable Cox proportional hazards regression models were used to test the correlations between PD-L1 expression and disease-free survival (DFS) and overall survival (OS). Results: The positive rates of SP142 (immune cell score, ICs≥1%) and 22C3 (combined positive score, CPS≥1) were 42.1%(109/259) and 41.3%(107/259) in TNBC tissues, respectively, with a total coincidence rate of 82.3%. The Kappa value of positive expression cases was 0.571 and the distribution difference of SP142 and 22C3 positive expression cases was statistically significant (P<0.001). The PD-L1 positive patients were less likely to have vascular invasion (P<0.05), but with higher histological grade and Ki-67 proliferation index (P<0.05). The recurrence/metastasis cases(8) of the patients with positive PD-L1 (SP142) was significantly lower than that of patients with negative PD-L1(SP142, 27, P=0.016). The positive expression of PD-L1 (SP142) patients were longer DFS (P=0.019). The OS of patients with positive PD-L1 (SP142) were longer than those with negative PD-L1 (SP142), but without significance (P=0.116). The positive expression of PD-L1 (22C3) was marginally associated with DFS and OS of patients (P>0.05). Conclusions: The expression of PD-L1 (22C3) is different from that of PD-L1 (SP142) in TNBC, and the two antibodies can't be interchangeable for each other in clinical tests. PD-L1 (SP142) status is an independent prognostic factor of DFS in TNBC. The DFS is significantly prolonged in patients with positive expression of PD-L1 (SP142).

Evaluating Mismatch Repair Status to Screen Clinical Advanced Breast Carcinomas for Immunotherapy: Experience From a Large Academic Institution.

BACKGROUND: Very few studies have investigated mismatch repair (MMR) deficiency in breast carcinoma (BC) in clinical setting. Given the recent approval of Pembrolizumab for solid tumors with MMR deficiency, we screened clinically advanced breast carcinoma patients for immunotherapy by examining their MMR status. PATIENTS AND METHODS: The cohort consisted of 163 clinical advanced BCs, including 5 primary, 14 locally recurrent, and 144 metastatic BCs. Immunohistochemistry (IHC) with anti-MMR proteins or next generation sequencing (NGS) to detect microsatellite instability was performed to evaluate MMR status. The relationship between MMR status and clinicopathologic characteristics was evaluated. RESULTS: Among 163 advanced BCs, 19 were hormone receptor (HR)-positive (≥ 10%)/HER2-negative, 17 were HER2+, and 127 were TNBCs/low HR-positive (< 10%). MMR status was evaluated by IHC in 131 cases and by NGS in 32 cases. Among all cases, only 1 case (0.6%) showed MMR deficiency. The case with MMR deficiency showed loss of MLH1 and PMS2 proteins, but no hypermethylation of MLH1 promoter. Sequencing analysis revealed MLH1 genetic alteration with a splice site mutation (208-1G > A), which results in disruption of the N-terminal ATPase-containing domain (amino acids 25-336). All 127 TNBCs/low HR-positive BCs showed preserved MMR. PD-L1 (SP142) testing was performed in 66 cases with 18 (27%) as positive and 48 (73%) as negative, and its expression showed no correlation with MMR status. CONCLUSION: MMR deficiency exists in an extremely low percentage of breast carcinomas, including TNBCs, suggesting a routine MMR testing to screen BC patients for immunotherapy may not be cost effective.

Fatty acid synthetase expression in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) has a relatively poor prognosis. Research has identified potential metabolic targets, including fatty acid metabolism, in TNBC. The absence of effective target therapies for TNBC led to exploration of the role of fatty acid synthetase (FASN) as a potential target for TNBC therapy. Here, we analyzed the expression of FASN, a representative lipid metabolism-related protein, and investigated the association between FASN expression and Ki-67 and the programmed death ligand 1 (PD-L1) biomarkers in TNBC. METHODS: Immunohistochemical expression of FASN was analyzed in 166 patients with TNBC. For analytical purposes, patients with 0-1+ FASN staining were grouped as low-grade FASN and patients with 2-3+ FASN staining as high-grade FASN. RESULTS: FASN expression was observed in 47.1% of TNBC patients. Low and high expression of FASN was identified in 75.9% and 24.1%, respectively, and no statistically significant difference was found in T category, N category, American Joint Committee on Cancer stage, or recurrence rate between the low and high-FASN expression groups. Ki-67 proliferation level was significantly different between the low and high-FASN expression groups. FASN expression was significantly related to Ki-67 as the level increased. There was no significant difference in PD-L1 positivity between the low- and high-FASN expression groups. CONCLUSIONS: We identified FASN expression in 166 TNBC patients. The Ki-67 proliferation index was positively correlated with FASN level, indicating higher proliferation activity as FASN increases. However, there was no statistical association with PD-L1 SP142, the currently FDA-approved assay, or FASN expression level.