It is shown that under certain conditions the iterative method suggested by the calculus of variations for calculating a cylindrically symmetric orientation distribution function (ODF) for rodlike particles strictly decreases the free energy at each full step. This monotonic behavior has strong implications for convergence of the sequence, or a subsequence, of the calculated ODFs. The result is valid not only for the reference system of hard core particles with indistinguishable ends, but also for free energy functions of similar type. The effect of an applied field is also permitted. Since the behavior of the iteration is intrinsic to the general form of the free energy function it applies to rod mixtures and may extend to a wider class of free energy functions. Outside the conditions that guarantee a monotonically decreasing free energy, we find that the iteration can fail to converge. For physically meaningful particle interactions, a natural iteration of the orientation distribution function decreases the free energy, F, at each step and, in practice, converges at a local free energy minimum.

BackgroundGastric carcinomas (GC) are aggressive malignancies, and only ~15% of patients respond to anti-programmed cell death (ligand) 1 (PD-(L)1) monotherapy. However, Epstein-Barr virus (EBV)-associated GCs (~5–10% of GCs) often harbor PD-L1 and PD-L2 chromosomal amplifications and robust CD8+ T cell infiltrates, and respond at a high rate to anti-PD-1. The current study compares the tumor immune microenvironments (TiMEs) of EBV+ versus EBV(−) GCs.MethodsOver 1000 cases of primary invasive GCs were screened to identify 25 treatment-naïve specimens for study (11 EBV+, 14 EBV(−)). Quantitative immunohistochemistry (IHC) was conducted for markers of immune cell subsets and co-regulatory molecules. Gene expression profiling (GEP) was performed on RNAs isolated from macrodissected areas of CD3+ T cell infiltrates abutting PD-L1+ stromal/tumor cells, using multiplex quantitative reverse transcriptase PCR for a panel of 122 candidate immune-related genes.ResultsIHC revealed that 17/25 GCs contained PD-L1+ stromal cells, with no significant difference between EBV+/- specimens; however, only 3/25 specimens (all EBV+) contained PD-L1+ tumor cells. CD8+ T cell densities were higher in EBV+ versus EBV(−) tumors (p=0.044). With GEP normalized to the pan-leukocyte markerPTPRC/CD45, EBV+ GCs overexpressedITGAE(CD103, marking intraepithelial T cells and a dendritic cell subset) and the interferon-inducible genesCXCL9andIDO1. In contrast, EBV(−) tumors overexpressed several functionally-related gene groups associated with myeloid cells (CD163,IL1A,NOS2, RIGI),immunosuppressive cytokines/chemokines (CXCL2,CXCR4,IL10, IL32),coinhibitory molecules (HAVCR2/TIM-3 andVSIR/VISTA), and adenosine pathway components (ENTPD1/CD39 andNT5E/CD73). Notably, compared with EBV+ GCs, EBV(−) GCs also overexpressed components of the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway associated with cancer-promoting inflammation, includingPTGS2/COX-2 (most highly upregulated gene, 32-fold, p=0.005); prostaglandin receptorsPTGER1(EP1; up 21-fold, p=0.015) andPTGER4(EP4; up twofold, p=0.022); and the major COX-2-inducing cytokineIL1B(up 11-fold, p=0.019). Consistent with these findings, COX-2 protein expression trended higher in EBV(−) versus EBV+ GCs (p=0.068).ConclusionsWhile certain markers of immunosuppression are found in the GC TiME regardless of EBV status, EBV(−) GCs, which are much more common than EBV+ GCs, overexpress components of the COX-2/PGE2 pathway. These findings provide novel insights into the immune microenvironments of EBV+ and EBV(−) GC, and offer potential targets to overcome resistance to anti-PD-(L)1 therapies.

Background Tumor regression following immune checkpoint blockade (ICB) is often associated with immune-related adverse events (irAEs), marked by inflammation in non-cancerous tissues. This study was undertaken to investigate the functional relationship between anti-tumor and anti-self immunity, to facilitate irAE management while promoting anti-tumor immunity. Methods Multiple biopsies from tumor and inflamed tissues were collected from a patient with melanoma experiencing both tumor regression and irAEs on ICB, who underwent rapid autopsy. Immune cells infiltrating melanoma lesions and inflamed normal tissues were subjected to gene expression profiling with multiplex qRT-PCR for 122 candidate genes. Subsequently, immunohistochemistry was conducted to assess the expression of 14 candidate markers of immune cell subsets and checkpoints. TCR-beta sequencing was used to explore T cell clonal repertoires across specimens. Results While genes involved in MHC I/II antigen presentation, IFN signaling, innate immunity and immunosuppression were abundantly expressed across specimens, irAE tissues over-expressed certain genes associated with immunosuppression ( CSF1R, IL10RA, IL27/EBI3, FOXP3, KLRG1, SOCS1, TGFB1 ), including those in the COX-2/PGE2 pathway ( IL1B, PTGER1/EP1 and PTGER4/EP4 ). Immunohistochemistry revealed similar proportions of immunosuppressive cell subsets and checkpoint molecules across samples. TCRseq did not indicate common TCR repertoires across tumor and inflammation sites, arguing against shared antigen recognition between anti-tumor and anti-self immunity in this patient. Conclusions This comprehensive study of a single patient with melanoma experiencing both tumor regression and irAEs on ICB explores the immune landscape across these tissues, revealing similarities between anti-tumor and anti-self immunity. Further, it highlights expression of the COX-2/PGE2 pathway, which is known to be immunosuppressive and potentially mediates ICB resistance. Ongoing clinical trials of COX-2/PGE2 pathway inhibitors targeting the major COX-2 inducer IL-1B, COX-2 itself, or the PGE2 receptors EP2 and EP4 present new opportunities to promote anti-tumor activity, but may also have the potential to enhance the severity of ICB-induced irAEs.

ARPE-19 cells are derived from adult human retinal pigment epithelium (RPE). The response of these cells to the stress of serum deprivation mimics some important processes relevant to age-related macular degeneration (AMD). Here we extend the characterization of this response using RNASeq and EGSEA gene set analysis of ARPE-19 cells over nine days of serum deprivation. This experiment confirmed the up-regulation of cholesterol and lipid-associated pathways that increase cholesterol levels in these cells. The gene expression analysis also identified other pathways relevant to AMD progression. There were significant changes in extracellular matrix gene expression, notably a switch from expression of collagen IV, a key component of Bruch’s membrane (part of the blood-retina barrier), to expression of a fibrosis-like collagen type I matrix. Changes in the expression profile of the extracellular matrix led to the discovery that amelotin is induced in AMD and is associated with the development of the calcium deposits seen in late-stage geographic atrophy. The transcriptional profiles of other pathways, including inflammation, complement, and coagulation, were also modified, consistent with immune response patterns seen in AMD. As previously noted, the cells resist apoptosis and autophagy but instead initiate a gene expression pattern characteristic of senescence, consistent with the maintenance of barrier function even as other aspects of RPE function are compromised. Other differentially regulated genes were identified that open new avenues for investigation. Our results suggest that ARPE-19 cells maintain significant stress responses characteristic of native RPE that are informative for AMD. As such, they provide a convenient system for discovery and for testing potential therapeutic interventions.

Supplementary Table S2: 1660 Illumina probes up-regulated at least 2-fold in PD-L1+ melanomas

Supplementary Table S1, Supplementary Table S3, Supplementary Table S5. Supplementary Table S1: Genes selected for multiplex qRT-PCR array Supplementary Table S3: Functionally annotated gene categories from DAVID analysis of whole genome microarray results comparing PD-L1+ to PD-L1(-) melanomas. Supplementary Table S5: Genes over-expressed in PD-L1+ vs. PD-L1(-) melanomas, assessed by qRT-PCR.

140 Illumina probes corresponding to138 unique genes differentially expressed in regressing (R) vs. progressing (P) cutaneous metastases based on whole genome microarray analysis (fold change magnitude {greater than or equal to}1.7, p value {less than or equal to}0.05 )

Abstract Background: Neoadjuvant anti-PD-1-based therapies have shown promise in several cancers, including Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. We have shown that on-treatment specimens from patients responding to anti-PD-1 are characterized histologically by exuberant proliferative fibrosis, neovascularization, and robust inflammation including diverse immune cell types. Understanding how the stroma participates in the process of tumor regression may open therapeutic opportunities by redirecting stromal remodeling in treatment-refractory tumors. Methods: To evaluate stromal changes in neoadjuvant treated MCC, 13 on-treatment FFPE tumor specimens from patients with resectable MCC who received anti-PD-1 for ~4 weeks before surgery (NCT02488759) were subjected to laser capture microdissection to collect stromal tissue and isolate RNA. We conducted multiplex qRT-PCR targeting 125 unique mRNAs representing TME-relevant immune cell subsets, immune checkpoint pathways, and immune-modulating transcription factors and cytokines, followed by whole transcriptome RNA sequencing. Differential gene expression was considered significant if the magnitude of the signed fold-change was ≥2 and the p-value was ≤0.1 (Benjamini-Hochberg adjusted for RNAseq). Gene set enrichment analysis (GSEA) was performed with R Topper. Pathologic response was scored on H&E staining as pathologic complete response (pCR, n=6), major pathologic response (MPR, ≤10% residual viable tumor [RVT]; n=4), or non-MPR/pCR (>10% RVT; n=3). Results: Stratifying by response status (6 pCR vs 3 non-MPR/pCR) demonstrated that pCR stroma in MCC is distinguished by upregulation of molecules associated with neovascularization (VEGFA, EGR2) and M1 and M2 macrophages (IL1A, ARG1) via qRT-PCR, and mesenchymal cells (fibroblasts, myocytes, adipocytes) and extracellular matrix formation via RNAseq. GSEA demonstrated a strong association of pCR with both myocyte and fibroblast differentiation and functional pathways, suggesting a role for myofibroblasts in tissue remodeling during anti-PD-1-mediated tumor regression. In contrast, the stroma of non-MPR/pCR tumors showed evidence for ongoing IFNg-mediated inflammation, as evidenced by upregulation of IFNg, IL-27, STAT1, CXCL10 and LAG-3 via qRT-PCR; these findings were supported by RNAseq, which also showed upregulation of genes associated with inflammation (e.g., PLXNC1, TRPV2, LYZ, LAIR1, HLA-A, B2M, CD4). Similar results were obtained when MPRs were grouped with pCRs (n=10) vs non-MPR/pCR (n=3). Conclusion: These findings demonstrate that improved response to anti-PD-1 therapy is associated with a shift in the tumor stroma from an inflammatory to a proliferative myofibroblastic phenotype, recapitulating the stages of normal wound healing. This suggests a potential functional role for myofibroblasts in that critical transition. Citation Format: Joel C. Sunshine, Tracee L. McMiller, Alyza Skaist, Yan Zhang, Alan Berger, Kornel Schuebel, Jennifer Meyers, Julie S. Deutsch, Aleksandra Ogurtsova, Elizabeth L. Engle, Leslie Cope, Janis M. Taube, Suzanne L. Topalian. Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2260.

Figure S1: Extraction of paraffin-embedded PD-L1+ RCC tissues for RNA isolation. Figure S2: Molecules previously found to be up-regulated in PD-L1+ vs. PD-L1(-) melanomas are not differentially expressed in PD-L1+ RCCs from patients with divergent clinical outcomes after anti-PD-1 therapy. Figure S3: Gene expression in RCC cell lines. Figure S4: In silico analyses of TCGA RCC data do not demonstrate significant associations between UGT1A6 gene expression and overall survival or clinical stage.

Figure S1, Figure S2, Figure S3. Supplementary Figure S1: Overlay of PD-L1 and LAG-3 IHC demonstrates close geographic association of these cell-surface markers in a melanoma lymph node metastasis. Supplementary Figure S2: Comparison of immunohistochemistry (IHC) and in situ hybridization (ISH) for LAG-3 expression in a melanoma lymph node metastasis. Supplementary Figure S3: IL-32-g activates CD3+ T cells and induces their expression of PD-L1.

Abstract Purpose: To explore factors associated with response and resistance to anti–PD-1 therapy, we analyzed multiple disease sites at autopsy in a patient with widely metastatic melanoma who had a heterogeneous response. Materials and Methods: Twenty-six melanoma specimens (four premortem, 22 postmortem) were subjected to whole exome sequencing. Candidate immunologic markers and gene expression were assessed in 10 cutaneous metastases showing response or progression during therapy. Results: The melanoma was driven by biallelic inactivation of NF1. All lesions had highly concordant mutational profiles and copy number alterations, indicating linear clonal evolution. Expression of candidate immunologic markers was similar in responding and progressing lesions. However, progressing cutaneous metastases were associated with overexpression of genes associated with extracellular matrix and neutrophil function. Conclusions: Although mutational and immunologic differences have been proposed as the primary determinants of heterogeneous response/resistance to targeted therapies and immunotherapies, respectively, differential lesional gene expression profiles may also dictate anti–PD-1 outcomes. Clin Cancer Res; 23(12); 3168–80. ©2017 AACR. See related commentary by Wilmott et al., p. 2921

Supplementary Table S4: DAVID clustering of 1660 Illumina probes up-regulated at least 2-fold in PD-L1+ melanomas

Supplementary Table S1: PD-L1+ RCC specimens used in this study. Supplementary Table S2: Genes differentially expressed in RCC based on whole genome microarray analysis, in patients responding or not to anti-PD-1 therapy (234 probe sets corresponding to 223 genes). Supplementary Table S3: Sixty genes included in custom multiplex qRT-PCR array to assess candidates from RCC whole genome microarray profiling.

Supplementary Materials and Methods

Whole exome sequencing data, including somatic variants, purities, private mutations, and predicted neoantigens.

Functionally annotated gene categories from DAVID analysis of Illumina probes differentially expressed between regressing vs. progressing melanoma metastases

Viruses are the second leading cause of cancer worldwide, and human papillomavirus (HPV)-associated head and neck cancers are increasing in incidence in the United States. HPV preferentially infects the crypts of the tonsils rather than the surface epithelium. The present study sought to characterize the unique microenvironment within the crypts to better understand the viral tropism of HPV to a lymphoid-rich organ. Lasercapture microdissection of distinct anatomic areas (crypts, surface epithelium, and germinal centers) of the tonsil, coupled with transcriptional analysis and multiparameter immunofluorescence staining demonstrated that the tonsillar crypts are enriched with myeloid populations that co-express multiple canonical and noncanonical immune checkpoints, including PD-L1, CTLA-4, HAVCR2 (TIM-3),ADORA2A, IDO1, BTLA, LGALS3, CDH1, CEACAM1, PVR, and C10orf54 (VISTA). The resident monocytes may foster a permissive microenvironment that facilitates HPV infection and persistence. Furthermore, the myeloid populations within HPVassociated tonsil cancers co-express the same immune checkpoints, providing insight into potential novel immunotherapeutic targets for HPV-associated head and neck cancers. (Am J Pathol 2021,191: 1774-1786; https://doi.org/10.1016/j.ajpath.2021.06.012)