Programmed death ligand 1 (PD-L1) is expressed in tumor cells and immune cells, and both have been associated with response to anti-PD-1 axis immunotherapy. Here, we examine the expression of PD-L1 to determine which cell type carries the predictive value of the test.Experimental Design: We measured the expression of PD-L1 in multiple immune cells with two platforms and confocal microscopy on three retrospective Yale NSCLC cohorts (425 nonimmunotherapy-treated cases and 62 pembrolizumab/nivolumab/atezolizumab-treated cases). The PD-L1 level was selectively measured in different immune cell subsets using two multiplexed quantitative immunofluorescence panels, including CD56 for natural killer cells, CD68 for macrophages, and CD8 for cytotoxic T cells.Results: PD-L1 was significantly higher in macrophages in both tumor and stromal compartment compared with other immune cells. Elevated PD-L1 in macrophages was correlated with high PD-L1 level in tumor as well as CD8 and CD68 level (P < 0.0001). High PD-L1 expression in macrophages was correlated with better overall survival (OS; P ¼ 0.036 by cell count/P ¼ 0.019 by molecular colocalization), while high PD-L1 expression in tumor cells was not.Conclusions: In nearly 500 non-small cell lung cancer (NSCLC) cases, the predominant immune cell type that expresses PD-L1 is CD68 þ macrophages. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor cells and infiltration by CD8 þ T cells, suggesting a connection between high PD-L1 and "hot" tumors. In anti-PD-1 axis therapytreated patients, high levels of PD-L1 expression in macrophages are associated with longer OS and may be responsible for the predictive effect of the marker.

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Assessment of tumor infiltrating lymphocytes (TILs) as a prognostic variable in melanoma has not seen broad adoption due to lack of standardization. Automation could represent a solution. Here, using open source software, we build an algorithm for image-based automated assessment of TILs on hematoxylin-eosin stained sections in melanoma. Using a retrospective collection of 641 melanoma patients comprising four independent cohorts; one training set (N = 227) and three validation cohorts (N = 137, N = 201, N = 76) from 2 institutions, we show that the automated TIL scoring algorithm separates patients into favorable and poor prognosis cohorts, where higher TILs scores were associated with favorable prognosis. In multivariable analyses, automated TIL scores show an independent association with disease-specific overall survival. Therefore, the open source, automated TIL scoring is an independent prognostic marker in melanoma. With further study, we believe that this algorithm could be useful to define a subset of patients that could potentially be spared immunotherapy.

◥Purpose: In both the IMpassion 130 trial in the metastatic setting and in Keynote 522 in the neoadjuvant setting, patients with triplenegative breast cancer (TNBC) showed benefit from PD-1 axis immunotherapy. Here, we assess PD-L1 expression on both tumor and immune cells using quantitative immunofluorescence to assess association with benefit from neoadjuvant durvalumab concurrent with chemotherapy in TNBC.Experimental Design: Pretreatment core needle biopsies (n ¼ 69) were obtained from patients who participated in a phase I/II clinical trial (NCT02489448). The final analysis included 45 patients [pathologic complete response (pCR) ¼ 18, non-pCR ¼ 27] due to technical issues and insufficient tissue. Slides were stained using a previously validated Ultivue DNA-based Ultimapper kit (CD8, CD68, PD-L1, Cytokeratin/Sox10, and Hoechst counterstain). The PD-L1 expression was analyzed by molecular compartmentalization without segmentation using AQUA software (version 3.2.2.1) in three tissue compartments including tumor (cytokeratinpositive cells), CD68 þ cells, and overall stroma.Results: In patients with pCR, PD-L1 expression was significantly higher in tumor cells, in CD68 þ cells and in the stroma compared with patients non-pCR. There was no difference in the amount of CD68 þ cells in the tumor or stromal compartments between cases with pCR and non-pCR.Conclusions: Expression of PD-L1 in tumor cells, immune cells in stroma, and colocalized with CD68 þ cells is associated with higher rates of pCR to durvalumab and chemotherapy in TNBC.

Introduction: CKLF like MARVEL transmembrane domain containing 6 (CMTM6) has been described as a programmed death ligand 1 (PD-L1) regulator at the protein level by modulating stability through ubiquitination. In this study, we describe the patterns of CMTM6 expression and assess its association with response to programmed cell death 1 pathway blockade in NSCLC. Methods: We used multiplexed quantitative immunofluorescence to determine the expression of CMTM6 and PD-L1 in 438 NSCLCs represented in tissue microarrays, including in two independent retrospective cohorts of immunotherapy-treated (n ¼ 69) and non-immunotherapytreated (n ¼ 258) patients and a third collection of EGFRand KRAS-genotyped tumors (n ¼ 111). Results: Tumor and stromal CMTM6 expression was detected in approximately 70% of NSCLCs. CMTM6 expression was not associated with clinical features or EGFR/KRAS mutational status and showed a modest correlation with T-cell infiltration (R 2 < 0.40). We found a significant correlation between CMTM6 and PD-L1, which was higher in the stroma (R 2 ¼ 0.51) than in tumor cells (R 2 ¼ 0.35). In our retrospective NSCLC cohort, neither CMTM6 nor PD-L1 expression alone significantly predicted immunotherapy outcomes. However, high CMTM6 and PD-L1 coexpression in the stromal and CD68 compartments (adjusted hazard ratio ¼ 0.38, p ¼ 0.03), but not in tumor cells (p ¼ 0.15), was significantly associated with longer overall survival in treated patients but was not observed in the absence of immunotherapy. Conclusion: This study supports the mechanistic role for CMTM6 in stabilization of PD-L1 in patient tumors and suggests that high coexpression of CMTM6 and PD-L1, particularly in stromal immune cells (macrophages), might identify the greatest benefit from programmed cell death 1 axis blockade in NSCLC.