PD-L1 is expressed in a percentage of lung cancer patients and those patients show increased likelihood of response to PD-1 axis therapies. However, the methods and assays for assessment of PD-L1 using immunohistochemistry are variable and PD-L1 expression appears to be highly heterogeneous. Here, we examine assay heterogeneity parameters toward the goal of determining variability of sampling and the variability due to pathologist-based reading of the immunohistochemistry slide. SP142, a rabbit monoclonal antibody, was used to detect PD-L1 by both chromogenic immunohistochemistry and quantitative immunofluorescenceusing a laboratory derived test. Five pathologists scored the percentage of PD-L1 positivity in tumor- and stromal immune cells of 35 resected non-small cell lung cancer cases, each represented on three separate blocks. An intraclass correlation coefficient of 94% agreement was seen among the pathologists for assessment of PD-L1 in tumor cells, but only 27% agreement was seen in stromal/immune cell PD-L1 expression. The block-to-block reproducibility of each pathologist’s score was 94% for tumor cells and 75% among stromal/immune cells. Lin’s concordance correlation coefficient between pathologists’ readings and the mean immunofluorescence score among blocks was 94% in tumor and 68% in stroma. Pathologists were highly concordant for PD-L1 tumor scoring, but not for stromal/immune cell scoring. Pathologist scores and immunofluorescence scores were concordant for tumor tissue, but not for stromal/immune cells. PD-L1 expression was similar among all 3 blocks from each tumor, indicating that staining of 1 block is enough to represent the entire tumor and that the spatial distribution of heterogeneity of expression of PD-L1 is within the area represented in a single block. Future studies are needed to determine the minimum representative tumor area for PD-L1 assessment for response to therapy.

BackgroundThe effects of neoadjuvant chemotherapy on immune markers remain largely unknown. The specific aim of this study was to assess stromal tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) protein expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy.MethodsUsing quantitative immunofluorescence, we investigated stromal TILs and PD-L1 protein expression in pre-treatment and residual breast cancer tissue from a Yale Cancer Center patient cohort of 58 patients diagnosed with breast cancer from 2003 to 2009 and treated with neoadjuvant chemotherapy. We compared the TIL count and PD-L1 status in paired pre-treatment and residual cancer tissues and correlated changes and baseline levels with survival.ResultsOf the 58 patients, 46 (79.3%) had hormone-positive and 34 (58.6%) had node-positive breast cancer. Eighty-six percent of residual cancer tissues had TIL infiltration and 17% had PD-L1 expression. There was a trend for higher TIL counts in postchemotherapy compared to prechemotherapy samples (p = 0.09). Increase in TIL count was associated with longer 5-year recurrence-free survival (p = 0.02, HR = 3.9, 95% CI = 1.179–15.39). PD-L1 expression (both stromal and tumor cells) was significantly lower in post-treatment samples (p = 0.001). Change in PD-L1 expression after therapy or TILs and PD-L1 expression in the posttreatment samples did not correlate with survival.ConclusionsIncrease in stromal TILs in residual cancer compared to pretreatment tissue is associated with improved recurrence-free survival. Despite a trend for increasing TIL counts, PD-L1 expression decreased in residual disease compared to pretreatment samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0884-8) contains supplementary material, which is available to authorized users.

Our aim was to examine the association of pretreatment tumor-infiltrating lymphocyte (TIL) count and PD-L1 levels with pathologic complete response (pCR) and assess immune marker changes following treatment in tumor specimens from the S0800 clinical trial, which randomized patients to bevacizumab + nab-paclitaxel, followed by doxorubicin/cyclophosphamide (AC) versus two control arms without bevacizumab (varying sequence of AC and nab-paclitaxel). TILs were assessed in 124 pre- and 62 posttreatment tissues (including 59 pairs). PD-L1 was assessed in 120 pre- and 43 posttreatment tissues (including 39 pairs) using the 22C3 antibody. Baseline and treatment-induced immune changes were correlated with pCR and survival using estrogen receptor (ER) and treatment-adjusted logistic and Cox regressions, respectively. At baseline, the mean TIL count was 17.4% (17% had zero TILs, 9% had ≥50% TILs). Posttreatment, mean TIL count decreased to 11% (5% had no TILs, 2% had >50% TILs). In paired samples, the mean TIL change was 15% decrease. Baseline PD-L1 was detected in 43% of cases ( = 5 in tumor cells, = 29 stroma, = 18 tumor + stroma). Posttreatment, PD-L1 expression was not significantly lower (33%). Higher baseline TIL count and PD-L1 positivity rate were associated with higher pCR rate even after adjustment for treatment and ER status ( = 0.018). There was no association between TIL counts, PD-L1 expression, and survival due to few events. In conclusion, TIL counts, but not PD-L1 expression, decreased significantly after treatment. Continued PD-L1 expression in some residual cancers raises the possibility that adjuvant immune checkpoint inhibitor therapy could improve survival in this patient population. .

Purpose: PD-1/PD-L1 axis inhibitors have been proven effective, especially in patients with tumors expressing Programmed Death Ligand 1 (PD-L1). Their clinical efficacy in patients with epidermal growth factor receptor (EGFR) activating mutations is still unclear, while KRAS mutations seem to be associated with good response. Experimental Design: We used multiplexed quantitative immunofluorescence (QIF) to investigate PD-L1 expression and to characterize Tumor Infiltrating Lymphocyte (TIL) populations and their activation status in over 150 Non-Small Cell Lung Cancer (NSCLC) patients with known mutation status. Results: PD-L1 expression was significantly lower in EGFR mutant compared to KRAS mutant and EGFR/KRAS Wild Type (WT) tumors. KRAS mutant tumors were more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis by TILs activation status revealed that EGFR mutants had a high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present, they were almost always active. Additionally, we found differences between EGFR mutation sites in CD8+ expression and TILs activation profile. Finally, activated EGFR correlated with increased PD-L1 expression in EGFR mutants but not in EGFR WT, while TIL activation was associated with higher PD-L1 only in EGFR/KRAS WT. Conclusions: Our findings demonstrate the unique immune profile of EGFR mutant tumors. The high frequency of inactive TILs could explain the low immunotherapy response rates in these patients, while PD-L1 as a predictive biomarker may reflect the constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD-L1 levels and TILs activation.

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