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Supplementary Table S1. Clinicopathologic characteristics of the cohort separated by death at the 5-year time point. Supplementary Table S2. Primary and secondary antibody information and TSA dye pairings for multiplex IF panel. Supplementary Table S3. Twenty highest ranking variables associating with overall survival by univariate Cox regression analysis. Supplementary Table S4. Density and proximity variables selected by least absolute shrinkage and selection operator (LASSO) methods using a 5-fold cross-validation. Supplementary Figure S1. Image cytometry to quantify immune cell biomarkers in metastatic melanoma. Supplementary Figure S2. The expression of PD-L1 on tumor cells and 'Other' cells is determined by their proximity to CD8+ cells. Supplementary Figure S3. Characterization of immune neighborhoods associated with overall survival. Supplementary Computational Methods. Survival and clinical statistical analysis - R markdown document.

Multiplex immunofluorescence (mIF) can detail spatial relationships and complex cell phenotypes in the tumor microenvironment (TME). However, the analysis and visualization of mIF data can be complex and time-consuming. Here, we used tumor specimens from 93 patients with metastatic melanoma to develop and validate a mIF data analysis pipeline using established flow cytometry workflows (image cytometry). Unlike flow cytometry, spatial information from the TME was conserved at single-cell resolution. A spatial uniform manifold approximation and projection (UMAP) was constructed using the image cytometry output. Spatial UMAP subtraction analysis (survivors vs. nonsurvivors at 5 years) was used to identify topographic and coexpression signatures with positive or negative prognostic impact. Cell densities and proportions identified by image cytometry showed strong correlations when compared with those obtained using gold-standard, digital pathology software (R2 > 0.8). The associated spatial UMAP highlighted “immune neighborhoods” and associated topographic immunoactive protein expression patterns. We found that PD-L1 and PD-1 expression intensity was spatially encoded—the highest PD-L1 expression intensity was observed on CD163+ cells in neighborhoods with high CD8+ cell density, and the highest PD-1 expression intensity was observed on CD8+ cells in neighborhoods with dense arrangements of tumor cells. Spatial UMAP subtraction analysis revealed numerous spatial clusters associated with clinical outcome. The variables represented in the key clusters from the unsupervised UMAP analysis were validated using established, supervised approaches. In conclusion, image cytometry and the spatial UMAPs presented herein are powerful tools for the visualization and interpretation of single-cell, spatially resolved mIF data and associated topographic biomarker development.

Purpose: PD-L1 expression in the pretreatment tumor microenvironment enriches for response to anti-PD-1/PD-L1 therapies. The purpose of this study was to quantitatively compare the performance of five monoclonal anti-PD-L1 antibodies used in recent landmark publications.Experimental Design: PD-L1 IHC was performed on 34 formalin-fixed paraffin-embedded archival melanoma samples using the 5H1, SP142, 28-8, 22C3, and SP263 clones. The percentage of total cells (including melanocytes and immune cells) demonstrating cell surface PD-L1 staining, as well as intensity measurements/H-scores, were assessed for each melanoma specimen using a computer-assisted platform. Staining properties were compared between antibodies.Results: Strong correlations were observed between the percentage of PD-L1(+) cells across all clones studied (R2 = 0.81–0.96). When present, discordant results were attributable to geographic heterogeneity of the melanoma tissue section rather than differences in PD-L1 antibody staining characteristics. PD-L1 intensity/H-scores strongly correlated with percentage of PD-L1(+) cells (R2 > 0.78, all clones).Conclusions: The 5H1, SP142, 28-8, 22C3, and SP263 clones all demonstrated similar performance characteristics when used in a standardized IHC assay on melanoma specimens. Reported differences in PD-L1 IHC assays using these antibodies are thus most likely due to assay characteristics beyond the antibody itself. Our findings also argue against the inclusion of an intensity/H-score in chromogenic PD-L1 IHC assays. Clin Cancer Res; 23(16); 4938–44. ©2017 AACR.

Supplemental Figures S1 Figure S2, Figure S3 and Table S1 and Table S2

Glioblastoma multiforme (GBM) is highly invasive and uniformly fatal, with median survival < 20 months after diagnosis even with the most aggressive treatment that includes surgery, radiation, and systemic chemotherapy. Cisplatin is a particularly potent chemotherapeutic agent, but its use to treat GBM is limited by severe systemic toxicity and inefficient penetration of brain tumor tissue even when it is placed directly in the brain within standard delivery systems. We describe the development of cisplatin-loaded nanoparticles that are small enough (70 nm in diameter) to move within the porous extracellular matrix between cells and that possess a dense polyethylene glycol (PEG) corona that prevents them from being trapped by adhesion as they move through the brain tumor parenchyma. As a result, these “brain penetrating nanoparticles” penetrate much deeper into brain tumor tissue compared to nanoparticles without a dense PEG corona following local administration by either manual injection or convection enhanced delivery. The nanoparticles also provide controlled release of cisplatin in effective concentrations to kill the tumor cells that they reach without causing toxicity-related deaths that were observed when cisplatin was infused into the brain without a delivery system. Median survival time of rats bearing orthotopic glioma was significantly enhanced when cisplatin was delivered in brain penetrating nano-particles (median survival not reached; 80% long-term survivors) compared to cisplatin in conventional un-PEGylated particles (median survival = 40 days), cisplatin alone (median survival = 12 days) or saline-treated controls (median survival = 28 days).

PD-L1 expression in the tumor immune microenvironment is recognized as both a prognostic and predictive biomarker in patients with cutaneous melanoma, a finding closely related to its adaptive (IFN-γ-mediated) mechanism of expression. Approximately 35% of cutaneous melanomas express PD-L1, however, the expression patterns, levels, and prevalence in rarer melanoma subtypes are not well described. We performed immunohistochemistry for PD-L1 and CD8 on 200 formalin-fixed paraffin-embedded specimens from patients with acral (n = 16), mucosal (n = 36), uveal (n = 103), and chronic sun-damaged (CSD) (n = 45) melanomas (24 lentigo maligna, 13 ‘mixed’ desmoplastic, and 8 ‘pure’ desmoplastic melanomas). CD8+ tumor-infiltrating lymphocyte (TIL) densities were characterized as mild, moderate, or severe, and their geographic association with PD-L1 expression was evaluated. Discrete lymphoid aggregates, the presence of a spindle cell morphology, and the relationship of these features with PD-L1 expression were assessed. PD-L1 expression was observed in 31% of acral melanomas, 44% of mucosal melanomas, 10% of uveal melanomas, and 62% of CSD melanomas (P

The discovery of powerful genetic targets has spurred clinical development of gene therapy approaches to treat patients with malignant brain tumors. However, lack of success in the clinic has been attributed to the inability of conventional gene vectors to achieve gene transfer throughout highly disseminated primary brain tumors. Here, we demonstrate ex vivo that small nanocomplexes composed of DNA condensed by a blend of biodegradable polymer, poly(β-amino ester) (PBAE), with PBAE conjugated with 5 kDa polyethylene glycol (PEG) molecules (PBAE-PEG) rapidly penetrate healthy brain parenchyma and orthotopic brain tumor tissues in rats. Rapid diffusion of these DNA-loaded nanocomplexes observed in fresh tissues ex vivo demonstrated that they avoided adhesive trapping in the brain owing to their dense PEG coating, which was critical to achieving widespread transgene expression throughout orthotopic rat brain tumors in vivo following administration by convection enhanced delivery. Transgene expression with the PBAE/PBAE-PEG blended nanocomplexes (DNA-loaded brain-penetrating nanocomplexes, or DNA-BPN) was uniform throughout the tumor core compared to nanocomplexes composed of DNA with PBAE only (DNA-loaded conventional nanocomplexes, or DNA-CN), and transgene expression reached beyond the tumor edge, where infiltrative cancer cells are found, only for the DNA-BPN formulation. Finally, DNA-BPN loaded with anti-cancer plasmid DNA provided significantly enhanced survival compared to the same plasmid DNA loaded in DNA-CN in two aggressive orthotopic brain tumor models in rats. These findings underscore the importance of achieving widespread delivery of therapeutic nucleic acids within brain tumors and provide a promising new delivery platform for localized gene therapy in the brain.

Astronomy accelerates tumor imaging Immunohistochemical stains for individual markers revolutionized diagnostic pathology decades ago but cannot capture enough information to accurately predict response to immunotherapy. Newer multiplex immunofluorescent technologies provide the potential to visualize the expression patterns of many functionally relevant molecules but present numerous challenges in accurate image analysis and data handling, particularly over large tumor areas. Drawing from the field of astronomy, in which petabytes of imaging data are routinely analyzed across a wide spectral range, Berry et al. developed a platform for multispectral imaging of whole-tumor sections with high-fidelity single-cell resolution. The resultant AstroPath platform was used to develop a multiplex immunofluorescent assay highly predictive of responses and outcomes for melanoma patients receiving immunotherapy. Science , aba2609, this issue p. eaba2609

Background: Multidimensional, spatially resolved analyses of immune and tumor cells within the TME of patients treated with checkpoint inhibitors will provide clinically translatable mechanistic insights and potentiate biomarker discovery. To achieve this goal, information from pathology specimens needs to be captured at a single cell level with high fidelity and in meaningfully sized cohorts. To date, efforts have been limited by inadequate tissue sampling and previously unrecognized errors in staining, imaging and data analysis. Here we describe the ‘AstroPath' platform, where strategies from the field of astronomy were adapted to study pathology specimens and generate large high quality mIF data. Methods: Potential error was identified and addressed at each stage of 6-plex (PD-1, PD-L1, FoxP3, CD163, CD8, tumor marker) mIF assay development. Whole slides from formalin-fixed paraffin embedded tissue specimens were stained with the optimized assay and imaged using a multispectral microscope (Vectra 3.0) with 20% overlap of high power fields (HPFs). The overlaps were used to quantify and correct optical lens distortion, HPF alignment, and illumination variation. Errors from cell segmentation algorithms, batch-to-batch staining variation, and HPF sampling were also addressed. The resultant mIF data were organized and analyzed using a large, relational database. Results: The optimized mIF assay captured equivalent signal compared to gold standard chromogenic immunohistochemistry and 2x more signal for PD-1, PD-L1 and FoxP3 compared to the manufacturer's recommended protocol. Errors and corrections for imaging included: pixel alignment error reduced from ~10 to Conclusion: Here we present an end-to-end pathology workflow with rigorous quality control for creating quantitative, spatially resolved mIF datasets using lessons derived from the field of astronomy. Such approaches will vastly improve standardization and scalability of mIF technologies, enabling cross-site comparisons and eventual clinical translation as biomarker discovery platforms or standard diagnostic tests. Citation Format: Sneha Berry, Nicolas Giraldo, Benjamin Green, Elizabeth Engle, Haiying Xu, Aleksandra Ogurtsova, Daphne Wang, Julie E. Stein, Peter Nguyen, Suzanne Topalian, Angelo DeMarzo, Drew M. Pardoll, Robert A. Anders, Tricia R. Cottrell, Alexander S. Szalay, Janis M. Taube. The ‘AstroPath' platform for spatially resolved, single cell analysis of the tumor microenvironment (TME) using multispectral immunofluorescence (mIF) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6584.

Multiplex IF (mIF) provides a detailed characterization of spatial relationships and complex cell phenotypes in the tumor microenvironment. However, the data-analysis and visualization is complex and time-consuming. Here, we developed a platform to analyze mIF data through flow cytometry workflows (image cytometry), while maintaining spatial information, and applied it to tissue microarrays of metastatic melanoma specimens (n=93; 6-plex mIF panel: PD-1, PD-L1, CD163, CD8, FoxP3, Sox10/S100). Then, we used a UMAP-based approach driven by cell-to-cell distances (rather than fluorescence intensity) to display and analyze geographic organization and cell interactions. Our pipeline provided equivalent results to the digital pathology gold standard with faster run times (5-fold reduction) and higher reproducibility. We identified key prognostic immune variables, including CD8PD1Low and CD8PD1Neg cells which associated with longer overall survival (OS, both p

Gene therapy is a promising strategy for the management of various neurological disorders that do not respond adequately to conventional therapeutics. The development of gene vectors with favorable safety profiles that can achieve uniform distribution and high-level transgene expression in the brain remains challenging. The rod-shaped, non-viral gene delivery platform based on poly-L-lysine (PLL) conjugated to a single segment of polyethylene glycol (PEG) has shown safe transfection in human nares and mouse brains in vivo. However, we have previously demonstrated that a denser PEG coating is required for rapid diffusion of nanoparticles in the brain extracellular space. Here, we engineered a densely PEGylated version of this platform based on PLL polymers conjugated to branched PEG via alkyne-azide cycloaddition. We found that the newly developed gene vectors rapidly diffused in the brain parenchyma, providing significantly improved vector distribution and overall transgene expression in vivo compared to the previously developed platform. These brain-penetrating DNA nanoparticles exhibited enhanced cellular uptake presumably due to their ellipsoidal morphology. By simultaneously improving delivery to target cells and subsequent transfection, our densely PEGylated PLL DNA nanoparticles can provide widespread, high levels of transgene expression, essential for effective targeting of highly disseminated brain diseases.

Convection enhanced delivery (CED) provides a powerful means to bypass the blood-brain barrier and drive widespread distribution of therapeutics in brain parenchyma away from the point of local administration. However, recent studies have detailed that the overall distribution of therapeutic nanoparticles (NP) following CED remains poor, due to tissue inhomogeneity and anatomical barriers present in the brain, which has limited its translational applicability. Using probe NP, we first demonstrate that a significantly improved brain distribution is achieved by infusing small, non-adhesive NP via CED in a hyperosmolar infusate solution. This multimodal delivery strategy minimizes the hindrance of NP diffusion imposed by the brain extracellular matrix and reduces NP confinement within the perivascular spaces. We further recapitulate the distributions achieved by CED of these probe NP using a most widely explored biodegradable polymer-based drug delivery NP. These findings provide a strategy to overcome several key limitations of CED that have been previously observed in clinical trials.

Purpose: PD-L1 expression in the pretreatment tumor microenvironment enriches for response to anti-PD-1/PD-L1 therapies. The purpose of this study was to quantitatively compare the performance of five monoclonal anti-PD-L1 antibodies used in recent landmark publications. Experimental Design: PD-L1 IHC was performed on 34 formalin-fixed paraffin-embedded archival melanoma samples using the 5H1, SP142, 28-8, 22C3, and SP263 clones. The percentage of total cells (including melanocytes and immune cells) demonstrating cell surface PD-L1 staining, as well as intensity measurements/H-scores, were assessed for each melanoma specimen using a computer-assisted platform. Staining properties were compared between antibodies. Results: Strong correlations were observed between the percentage of PD-L1(+) cells across all clones studied (R2 = 0.81–0.96). When present, discordant results were attributable to geographic heterogeneity of the melanoma tissue section rather than differences in PD-L1 antibody staining characteristics. PD-L1 intensity/H-scores strongly correlated with percentage of PD-L1(+) cells (R2 > 0.78, all clones). Conclusions: The 5H1, SP142, 28-8, 22C3, and SP263 clones all demonstrated similar performance characteristics when used in a standardized IHC assay on melanoma specimens. Reported differences in PD-L1 IHC assays using these antibodies are thus most likely due to assay characteristics beyond the antibody itself. Our findings also argue against the inclusion of an intensity/H-score in chromogenic PD-L1 IHC assays. Clin Cancer Res; 23(16); 4938–44. ©2017 AACR.

Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic non-biodegradable systems. Using a state-of-the-art biodegradable polymer, poly(β-amino ester), we engineered colloidally stable sub-100 nm DNA nanoparticles coated with a non-adhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles were able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases.

We recently reported a 56% objective response rate in patients with advanced MCC receiving pembrolizumab (anti-PD-1) as first-line therapy. However, a tumor biomarker predicting clinical response was not defined. The purpose of this study was to determine potential associations of anti-PD-1 response (RECIST 1.1, analysis 8/1/16) and the presence of MCPyV, with the density and distribution of CD8+, PD-1+ and PD-L1+ cell populations in the tumor microenvironment (TME). Pretreatment FFPE tumor specimens were stained for CD8 (n=23) and PD-L1 (n=16, 22C3 assay) with immunohistochemistry (IHC). Immunofluorescence (IF) was used to detect PD-1 (n=16). Intratumoral (IT), peritumoral (PT, 100 um zone) and total (PT+IT) densities of PD-1+ and CD8+ immune cells (IC) were determined with digital image analysis. PD-L1+ cases had >1% tumor cells (TC) expressing PD-L1. Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ cells in the IT, PT, and IT+PT regions when compared to non-responders (Mann-Whitney test, p-value=0.03, 0.06, 0.03, respectively). There was no significant association of response with CD8+ IC densities (IT, PT or total) or TC PD-L1 expression. Similarly, when we quantified the number of PD-1+ IC located within 15 um from a PD-L1+ cell (TC or IC), an association was observed between PD-1 and PD-L1 proximity and clinical response (Mann-Whitney test, p-value =0.04), but not CD8 and PD-L1 proximity. Because viral neoantigens can elicit a strong immune response, we also studied these TME factors for their potential associations with MCPyV. We found the presence of virus was significantly associated with increased densities of PT CD8+ cells (Mann-Whitney test, p-value=0.008) and TC PD-L1 expression (Fisher's test, p-value=0.04), but not PD-1+ IC. A more extensive multiplex IF panel (CD8, PD-1, PD-L1, CD68, FoxP3, NSE) analysis was performed on select pre-treatment specimens using the Vectra multispectral imaging system (Perkin Elmer) to explore this divergence. We found that only ~60% of observed total PD-1 expression was displayed by CD8+ cells. Although preliminary, our results suggest a relationship between PD-1+ cells and response to anti-PD-1 therapy and highlight that lymphocyte subsets other than CD8+ T-cells may contribute to the observed response. Further studies in larger cohorts are needed to validate these results. Citation Format: Nicolas A. Giraldo, Genevieve J. Kaunitz, Tricia R. Cottrell, Sneha Berry, Joel C. Sunshine, Peter Nguyen, Haiying Xu, Aleksandra Orgutsova, Candice D. Church, Natalie J. Miller, Jennifer H. Yearley, Evan J. Lipson, Ludmila Danilova, Paul T. Nghiem, Suzanne L. Topalian, Janis M. Taube. The differential association of PD-1, PD-L1, and CD8+ cells with response to pembrolizumab and presence of Merkel cell polyomavirus (MCPyV) in patients with Merkel cell carcinoma (MCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2017-662

Gene delivery to the central nervous system (CNS) has potential as a means for treating numerous debilitating neurological diseases. Non-viral gene vector platforms are tailorable and can overcome key limitations intrinsic to virus-mediated delivery; however, lack of clinical efficacy with non-viral systems to date may be attributed to limited gene vector dispersion and transfection in vivo. We show that the brain extracellular matrix strongly limits penetration of polymer-based gene vector nanoparticles through the brain parenchyma, even when they are very small (

Gene therapy constitutes an attractive approach for curing a variety of CNS diseases, including glioblastoma. Multiple studies have been carried out focusing on improving the total level and distribution of gene expression throughout the brain tissue. Viral gene delivery, though relatively efficient, has been limited by immunogenicity, low packaging capacity and difficulties in scale-up. Non-viral gene vectors offer an alternative strategy but are limited in their ability to generate high-level, widespread therapeutic effect. The nanoporous and electrostatically charged extracellular space found between healthy or tumor cells constitutes a steric and adhesive barrier, which hampers widespread distribution of gene vectors in the brain, regardless of administration method, thereby limiting gene transfer to target cells [1]. Indeed, following convention enhanced delivery (CED) conventional gene vectors are confined to the point of administration, limiting the clinical success of gene therapy for glioblastoma [2]. We developed a strategy to formulate small, stable and densely coated cationic polymer-based gene vectors that have the physicochemical characteristics required to overcome this ‘brain tissue barrier’. Using high resolution multiple particle tracking, we assessed the diffusion rates of gene vectors in freshly extracted ex vivo healthy and tumor rodent brain tissues. We thoroughly characterized their toxicity, cell uptake and transfection efficacy in vitro. Finally, we analyzed the ability of these vectors to rapidly penetrate and distribute throughout the brain tissue in vivo, thus reaching and transfecting cells over a large volume following CED. 1.Nance, E.A., et al., Sci Transl Med, 2012. 2.Voges, J., et al., Ann Neurol, 2003.

Meeting abstracts Immunohistochemical detection of PD-L1 may be used in the future as a biomarker to help select an immunotherapeutic regimen for patients with advanced melanoma. For example, patients whose tumors are PD-L1+ may receive anti-PD-1 monotherapy, and those whose tumors are PD-L1(-) may

Oncogenic driver mutations in several tumor types promote constitutive PD-L1 expression, a crucial ligand in PD-1-mediated tumor immune escape. Our studies in melanoma suggest a different mechanism–one of “adaptive immune resistance” in which PD-L1 expression is primarily driven by cytokine induction and is independent of BRAF mutational status.