Immune cell subsets in interface cutaneous immune-related adverse events associated with anti-PD-1 therapy resemble acute graft versus host disease more than lichen planus.

BACKGROUND: Checkpoint immunotherapy is frequently associated with cutaneous immune-related adverse events (cirAEs), and among those, the most common subtype shows interface reaction patterns that have been likened to lichen planus (LP); however, cutaneous acute graft versus host disease (aGVHD) may be a closer histopathologic comparator. We used quantitative pathology to compare the immunologic composition of anti-PD-1-associated interface reactions to LP and aGVHD to assess for similarities and differences between these cutaneous eruptions. METHODS: Immunohistochemistry for CD4, CD8, CD68, PD-1, and PD-L1 was performed on formalin-fixed paraffin-embedded tissue from patients with anti-PD-1 interface cirAEs (n = 4), LP (n = 9), or aGVHD (n = 5). Densities of immune cell subsets expressing each marker were quantified using the HALO image analysis immune cell module. Plasma cell and eosinophil density were quantified on routine H&E slides. RESULTS: Specimens from patients with anti-PD-1 interface cirAEs showed equivalent total cell densities and immune cell composition to those with aGVHD. Patients with LP showed higher total immune cell infiltration, higher absolute T-cell densities, increased CD8 proportion, and reduced histiocytic component. The cases with the highest plasma cell counts were all anti-PD-1 interface cirAEs and aGVHD. CONCLUSION: The composition of immune cell subsets in anti-PD-1 interface cirAEs more closely resembles the immune response seen in aGVHD than LP within our cohort. This warrants a closer look via advanced analytics and may have implications for shared pathogenesis and potential treatment options.

Tumor MHC Class I Expression Associates with Intralesional IL2 Response in Melanoma.

Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1-LAG-3-TIM-3-). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as "extreme responders") were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.

Neoadjuvant Nivolumab in Patients with High-risk Nonmetastatic Renal Cell Carcinoma.

Neoadjuvant immune checkpoint blockade represents a novel approach for potentially decreasing the risk of recurrence in patients with nonmetastatic renal cell carcinoma (RCC). In this early phase clincal tiral, we evaluated the safety and tolerability of neoadjuvant treatment with the programmed cell death protein 1 (PD-1) inhibitor nivolumab in patients with nonmetastatic high-risk RCC. Nonprimary endpoints included objective radiographic tumor response rate, immune-related pathologic response rate, quality of life alterations, and metastasis-free and overall survival. In total, 17 patients were enrolled in this study and underwent surgery without a delay after receiving three every-2-wk doses of neoadjuvant nivolumab. Adverse events (AEs) of any grade occurred in 14 (82.4%) patients, with two (11.8%) experiencing grade 3 events. Ten (58.8%) patients experienced an AE of any grade potentially attributable to nivolumab (all grade 1-2), and no grade 4-5 AEs occurred regardless of treatment attribution. The most common AEs were grade 1 fatigue (41.2%), grade 1 pruritis (29.4%), and grade 1 rash (29.4%). All evaluable patients had stable disease as per established radiographic criteria, with one (6.7%) demonstrating features of an immune-related pathologic response. Quality of life remained stable during treatment, with improvements relative to baseline noted at ≥6 mo postoperatively. Metastasis-free survival and overall survival were 85.1% and 100% at 2 yr, respectively. PATIENT SUMMARY: In this study, we evaluated the safety and tolerability of preoperative administration of three doses of the immune checkpoint inhibitor nivolumab in patients with clinically localized high-risk renal cell carcinoma. We demonstrated the safety of this approach and found that, although most patients will not experience a radiographic response to treatment, a subset may have features of an immune-related pathologic response.

Spatial UMAP and Image Cytometry for Topographic Immuno-oncology Biomarker Discovery.

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.

Neoadjuvant nivolumab for patients with resectable HPV-positive and HPV-negative squamous cell carcinomas of the head and neck in the CheckMate 358 trial.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are common malignancies caused by carcinogens, including tobacco and alcohol, or infection with human papillomavirus (HPV). Immune checkpoint inhibitors targeting the programmed cell death 1 (PD-1) pathway are effective against unresectable recurrent/metastatic HNSCC. Here, we explored the safety and efficacy of anti-PD-1 therapy in at-risk resectable HPV-positive and HPV-negative HNSCC in the neoadjuvant setting. METHODS: The phase I/II CheckMate 358 trial in virus-associated cancers assessed neoadjuvant nivolumab in patients with previously untreated, resectable HPV-positive or HPV-negative HNSCC. Patients received nivolumab 240 mg intravenously on days 1 and 15, with surgery planned by day 29. Safety/tolerability (primary endpoint) was assessed by monitoring adverse events (AEs) and surgical delays. Radiographic response was measured before surgery using RECIST v1.1, adapted for a single post-nivolumab evaluation. Pathologic specimens were examined for treatment response using immune-based criteria. RESULTS: From November 2015 to December 2017, 52 patients with AJCC (seventh edition) stage III-IV resectable HNSCC received neoadjuvant nivolumab (26 HPV-positive, 26 HPV-negative). Any-grade treatment-related AEs (TRAEs) occurred in 19 patients (73.1%) and 14 patients (53.8%) in the HPV-positive and HPV-negative cohorts, respectively; grade 3-4 TRAEs occurred in five (19.2%) and three patients (11.5%), respectively. No patient had a protocol-defined TRAE-related surgical delay (>4 weeks). Thirty-eight patients were reported as undergoing complete surgical resection, 10 had a planned post-nivolumab biopsy instead of definitive surgery due to a protocol misinterpretation, and four did not undergo surgery or biopsy, including two with tumor progression. Radiographic response rates in 49 evaluable patients were 12.0% and 8.3% in the HPV-positive and HPV-negative cohorts, respectively. There were no complete pathologic responses by site or central review in operated patients. Among 17 centrally evaluable HPV-positive tumors, one (5.9%) achieved major pathological response and three (17.6%) achieved partial pathologic response (pPR); among 17 centrally evaluable HPV-negative tumors, one (5.9%) achieved pPR. CONCLUSIONS: Neoadjuvant nivolumab was generally safe and induced pathologic regressions in HPV-positive (23.5%) and HPV-negative (5.9%) tumors. Combinatorial neoadjuvant treatment regimens, and continued postoperative therapy for high-risk tumors, are warranted in future trials to enhance the efficacy of this approach. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02488759; https://clinicaltrials.gov/ct2/show/NCT02488759.

Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD-1 blockade.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163+PD-L1- myeloid cells and CD8+FoxP3+PD-1low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.

Fatal SARS-CoV-2 Inflammatory Syndrome and Myocarditis in an Adolescent: A Case Report.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an entity in children initially characterized by milder case presentations and better prognoses as compared with adults. Recent reports, however, raise concern for a new hyperinflammatory entity in a subset of pediatric COVID-19 patients. METHODS: We report a fatal case of confirmed COVID-19 with hyperinflammatory features concerning for both multi-inflammatory syndrome in children (MIS-C) and primary COVID-19. RESULTS: This case highlights the ambiguity in distinguishing between these two entities in a subset of pediatric patients with COVID-19-related disease and the rapid decompensation these patients may experience. CONCLUSIONS: Appropriate clinical suspicion is necessary for both acute disease and MIS-C. SARS-CoV-2 serologic tests obtained early in the diagnostic process may help to narrow down the differential but does not distinguish between acute COVID-19 and MIS-C. Better understanding of the hyperinflammatory changes associated with MIS-C and acute COVID-19 in children will help delineate the roles for therapies, particularly if there is a hybrid phenotype occurring in adolescents.

Characterization of the tumor immune microenvironment in human papillomavirus-positive and -negative head and neck squamous cell carcinomas.

Approximately 15% of advanced head and neck squamous cell carcinomas (HNSCC) respond to anti-PD-(L)1 monotherapies. Tumor PD-L1 expression and human papillomavirus (HPV) status have been proposed as biomarkers to identify patients likely to benefit from these treatments. We aimed to understand the potential immune effects of HPV in HNSCC and to characterize additional potentially targetable immune-regulatory pathways in primary, treatment-naïve tumors. CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L2, LAG-3, IDO-1, and GITR cell densities were determined in 27 HNSCC specimens. IHC for PD-L1 assessed percentage of positive tumor cells and immune cells separately or as a combined positive score (CPS), and whether PD-L1 was expressed in an adaptive or constitutive pattern (i.e., PD-L1+ tumor cells juxtaposed to TILs or in the absence of TILs, respectively). HPV testing with p16 IHC was confirmed by HPV genotyping. When compared to HPV(-) tumors (n = 14), HPV+ tumors (n = 13) contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells (P < 0.02), and there was a trend towards increased density of FoxP3 + cells. PD-L1 expression patterns did not vary by tumor viral status, suggesting possible heterogeneous mechanisms driving constitutive vs adaptive PD-L1 expression patterns in HNSCC. IDO-1 expression was abundant (> 500 IDO-1+ cells/mm2 in 17/27 specimens) and was found on tumor cells as well as immune cells in 12/27 (44%) cases (range 5-80% tumor cells+). Notably, the studied markers varied on a per-patient basis and were not always related to the degree of T cell infiltration. These findings may inform therapeutic co-targeting strategies and raise consideration for a personalized treatment approach.

Neoadjuvant Nivolumab for Patients With Resectable Merkel Cell Carcinoma in the CheckMate 358 Trial.

PURPOSE: Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer commonly driven by the Merkel cell polyomavirus (MCPyV). The programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) immunosuppressive pathway is often upregulated in MCC, and advanced metastatic MCC frequently responds to PD-1 blockade. We report what we believe to be the first trial of anti-PD-1 in the neoadjuvant setting for resectable MCC. METHODS: In the phase I/II CheckMate 358 study of virus-associated cancer types, patients with resectable MCC received nivolumab 240 mg intravenously on days 1 and 15. Surgery was planned on day 29. Tumor regression was assessed radiographically and microscopically. Tumor MCPyV status, PD-L1 expression, and tumor mutational burden (TMB) were assessed in pretreatment tumor biopsies. RESULTS: Thirty-nine patients with American Joint Committee on Cancer stage IIA-IV resectable MCC received ≥ 1 nivolumab dose. Three patients (7.7%) did not undergo surgery because of tumor progression (n = 1) or adverse events (n = 2). Any-grade treatment-related adverse events occurred in 18 patients (46.2%), and grade 3-4 events in 3 patients (7.7%), with no unexpected toxicities. Among 36 patients who underwent surgery, 17 (47.2%) achieved a pathologic complete response (pCR). Among 33 radiographically evaluable patients who underwent surgery, 18 (54.5%) had tumor reductions ≥ 30%. Responses were observed regardless of tumor MCPyV, PD-L1, or TMB status. At a median follow-up of 20.3 months, median recurrence-free survival (RFS) and overall survival were not reached. RFS significantly correlated with pCR and radiographic response at the time of surgery. No patient with a pCR had tumor relapse during observation. CONCLUSION: Nivolumab administered approximately 4 weeks before surgery in MCC was generally tolerable and induced pCRs and radiographic tumor regressions in approximately one half of treated patients. These early markers of response significantly predicted improved RFS. Additional investigation of these promising findings is warranted.

Pan-Tumor Pathologic Scoring of Response to PD-(L)1 Blockade.

PURPOSE: Pathologic response assessment of tumor specimens from patients receiving systemic treatment provides an early indication of therapeutic efficacy and predicts long-term survival. Grading systems for pathologic response were first developed for chemotherapy in select tumor types. Immunotherapeutic agents have a mechanism of action distinct from chemotherapy and are being used across a broad array of tumor types. A standardized, universal scoring system for pathologic response that encompasses features characteristic for immunotherapy and spans tumor types is needed. EXPERIMENTAL DESIGN: Hematoxylin and eosin-stained slides from neoadjuvant surgical resections and on-treatment biopsies were assessed for features of immune-related pathologic response (irPR). A total of 258 specimens from patients with 11 tumor types as part of ongoing clinical trials for anti-PD-(L)1 were evaluated. An additional 98 specimens from patients receiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types. RESULTS: Common irPR features (immune activation, cell death, tissue repair, and regression bed) were present in all tumor types reviewed, including melanoma, non-small cell lung, head and neck squamous cell, Merkel cell, and renal cell carcinoma, among others. Features were consistent across primary tumors, lymph nodes, and distant metastases. Specimens from patients treated with anti-PD-(L)1 in combination with another agent also exhibited irPR features. CONCLUSIONS: irPR features are consistent across tumor types and treatment settings. Standardized, pan-tumor irPR criteria (irPRC) are defined and associated specimen-handling considerations are described. Future, prospective studies are merited to validate irPRC in larger datasets and to associate pathologic features with long-term patient outcomes.

Interleukin-36γ-producing macrophages drive IL-17-mediated fibrosis.

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)-4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17-dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.

Comparison of Biomarker Modalities for Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Review and Meta-analysis.

IMPORTANCE: PD-L1 (programmed cell death ligand 1) immunohistochemistry (IHC), tumor mutational burden (TMB), gene expression profiling (GEP), and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) assays have been used to assess pretreatment tumor tissue to predict response to anti-PD-1/PD-L1 therapies. However, the relative diagnostic performance of these modalities has yet to be established. OBJECTIVE: To compare studies that assessed the diagnostic accuracy of PD-L1 IHC, TMB, GEP, and mIHC/IF in predicting response to anti-PD-1/PD-L1 therapy. EVIDENCE REVIEW: A search of PubMed (from inception to June 2018) and 2013 to 2018 annual meeting abstracts from the American Association for Cancer Research, American Society of Clinical Oncology, European Society for Medical Oncology, and Society for Immunotherapy of Cancer was conducted to identify studies that examined the use of PD-L1 IHC, TMB, GEP, and mIHC/IF assays to determine objective response to anti-PD-1/PD-L1 therapy. For PD-L1 IHC, only clinical trials that resulted in US Food and Drug Administration approval of indications for anti-PD-1/PD-L1 were included. Studies combining more than 1 modality were also included. Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines were followed. Two reviewers independently extracted the clinical outcomes and test results for each individual study. MAIN OUTCOMES AND MEASURES: Summary receiver operating characteristic (sROC) curves; their associated area under the curve (AUC); and pooled sensitivity, specificity, positive and negative predictive values (PPV, NPV), and positive and negative likelihood ratios (LR+ and LR-) for each assay modality. RESULTS: Tumor specimens representing over 10 different solid tumor types in 8135 patients were assayed, and the results were correlated with anti-PD-1/PD-L1 response. When each modality was evaluated with sROC curves, mIHC/IF had a significantly higher AUC (0.79) compared with PD-L1 IHC (AUC, 0.65, P < .001), GEP (AUC, 0.65, P = .003), and TMB (AUC, 0.69, P = .049). When multiple different modalities were combined such as PD-L1 IHC and/or GEP + TMB, the AUC drew nearer to that of mIHC/IF (0.74). All modalities demonstrated comparable NPV and LR-, whereas mIHC/IF demonstrated higher PPV (0.63) and LR+ (2.86) than the other approaches. CONCLUSIONS AND RELEVANCE: In this meta-analysis, tumor mutational burden, PD-L1 IHC, and GEP demonstrated comparable AUCs in predicting response to anti-PD-1/PD-L1 treatment. Multiplex immunohistochemistry/IF and multimodality biomarker strategies appear to be associated with improved performance over PD-L1 IHC, TMB, or GEP alone. Further studies with mIHC/IF and composite approaches with a larger number of patients will be required to confirm these findings. Additional study is also required to determine the most predictive analyte combinations and to determine whether biomarker modality performance varies by tumor type.

Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.

BACKGROUND: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified. METHODS: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1. RESULTS: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 µm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1. CONCLUSIONS: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.

Anti-TNF therapy-induced lupus erythematosus-like syndrome in a patient treated with adalimumab for cutaneous psoriasis.

A 44-year-old woman with cutaneous psoriasis and no history of joint involvement recently treated with adalimumab was admitted to the inpatient Internal Medicine service for uncontrolled, severe joint pain so debilitating that it limited her activities of daily living and prevented her from working as a medical technologist. She had stopped taking adalimumab 3 weeks prior to presentation after receiving approximately 2.5 months of therapy for cutaneous psoriasis unresponsive to trials of topical steroids and methotrexate. Antinuclear antibody and anti-double-stranded DNA antibodies were positive at high titres. She received a course of oral corticosteroids with improvement in her symptoms.