Multiparameter Immunofluorescence Research Articles

Aberrant Activation of Wound-Healing Programs within the Metastatic Niche Facilitates Lung Colonization by Osteosarcoma Cells.

Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. The purpose of this study is to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms underlying osteosarcoma lung metastatic niche formation. Using single-cell RNA sequencing, we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multiparameter immunofluorescence and spatial transcriptomics. Based on these findings, we evaluated the ability of nintedanib, a kinase inhibitor used to treat patients with pulmonary fibrosis, to impair metastasis progression in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Single-nucleus and spatial transcriptomics were used to perform molecular pharmacodynamic studies that define the effects of nintedanib on tumor and nontumor cells within the metastatic microenvironment. Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. Single-cell RNA sequencing demonstrated that the surrounding lung stroma adopts a chronic, nonresolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, the metastasis-associated lung demonstrated marked fibrosis, likely because of the accumulation of pathogenic, profibrotic, partially differentiated epithelial intermediates and macrophages. Our data demonstrated that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis. Fibrosis represents a targetable vulnerability to block the progression of osteosarcoma lung metastasis. Our data support a model wherein interactions between osteosarcoma cells and epithelial cells create a prometastatic niche by inducing tumor deposition of extracellular matrix proteins such as fibronectin that is disrupted by the antifibrotic tyrosine kinase inhibitor (TKI) nintedanib. Our data shed light on the non-cell-autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKI on metastases in animal models.

42-parameter mass cytometry panel to assess cellular and functional phenotypes of leukocytes in bronchoalveolar lavage of Rhesus macaque.

This Optimized Multiparameter Immunofluorescence Panel (OMIP) reports on the development of a mass cytometry panel for broad immunophenotyping of leukocytes from bronchoalveolar lavage from rhesus macaques. Using this panel, we were able to identify myeloid populations such as macrophages, neutrophils, monocytes, myeloid and plasmacytoid DCs, basophils and lymphoid cell lineages including B cells, natural killer (NK) cells, mucosal associated invariant T (MAIT) cells, γδ T cells, CD4 T cells, CD8 β T cells, CD8 T cells, and innate lymphoid cells (ILCs). We also included markers for defining memory, differentiation (CCR7, CD28, CD45RA), homing potential (CXCR3), cytotoxic potential (perforin, granzyme B, granzyme K), cell activation/differentiation (HLA-DR, CD69, IgD) and effector function (CD154, IFN-γ, TNF, IL-2, IL-17A, IL-6, IL-1β, CCL4 and CD107a). This panel was optimized on cryopreserved, bronchoalveolar lavage and splenocytes collected from rhesus macaques. The antibodies selected in this panel are human-specific antibodies that have been shown to cross-react with non-human primates except for CD45 clone D058-1283 which is specific for non-human primates.

Aberrant activation of wound healing programs within the metastatic niche facilitates lung colonization by osteosarcoma cells.

Lung metastasis is responsible for nearly all deaths caused by osteosarcoma, the most common pediatric bone tumor. How malignant bone cells coerce the lung microenvironment to support metastatic growth is unclear. The purpose of this study is to identify metastasis-specific therapeutic vulnerabilities by delineating the cellular and molecular mechanisms underlying osteosarcoma lung metastatic niche formation. Using single-cell transcriptomics (scRNA-seq), we characterized genome- and tissue-wide molecular changes induced within lung tissues by disseminated osteosarcoma cells in both immunocompetent murine models of metastasis and patient samples. We confirmed transcriptomic findings at the protein level and determined spatial relationships with multi-parameter immunofluorescence and spatial transcriptomics. Based on these findings, we evaluated the ability of nintedanib, a kinase inhibitor used to treat patients with pulmonary fibrosis, to impair metastasis progression in both immunocompetent murine osteosarcoma and immunodeficient human xenograft models. Single-nucleus and spatial transcriptomics was used to perform molecular pharmacodynamic studies that define the effects of nintedanib on tumor and non-tumor cells within the metastatic microenvironment. Osteosarcoma cells induced acute alveolar epithelial injury upon lung dissemination. scRNA-seq demonstrated that the surrounding lung stroma adopts a chronic, non-resolving wound-healing phenotype similar to that seen in other models of lung injury. Accordingly, metastasis-associated lung demonstrated marked fibrosis, likely due to the accumulation of pathogenic, pro-fibrotic, partially differentiated epithelial intermediates and macrophages. Our data demonstrated that nintedanib prevented metastatic progression in multiple murine and human xenograft models by inhibiting osteosarcoma-induced fibrosis. Fibrosis represents a targetable vulnerability to block the progression of osteosarcoma lung metastasis. Our data support a model wherein interactions between osteosarcoma cells and epithelial cells create a pro-metastatic niche by inducing tumor deposition of extracellular matrix proteins such as fibronectin that is disrupted by the anti-fibrotic TKI nintedanib. Our data shed light on the non-cell autonomous effects of TKIs on metastasis and provide a roadmap for using single-cell and spatial transcriptomics to define the mechanism of action of TKI on metastases in animal models.

T-cell infiltrate intensity is associated with delayed response to treatment in late acute cellular rejection in pediatric liver transplant recipients.

Late acute cellular rejection (ACR) is associated with donor-specific antibodies (DSA) development, chronic rejection, and allograft loss. However, accurate predictors of late ACR treatment response are lacking. ACR is primarily T-cell mediated, yet B cells and plasma cells (PC) also infiltrate the portal areas during late ACR. To test the hypothesis that the inflammatory milieu is associated with delayed response (DR) to rejection therapy, we performed a single-center retrospective case-control study of pediatric late liver ACR using multiparameter immunofluorescence for CD4, CD8, CD68, CD20, and CD138 to identify immune cell subpopulations. Pediatric liver transplant recipients transplanted at <17 years of age and treated for biopsy-proven late ACR between January 2014 and 2019 were stratified into rapid response (RR) and DR based on alanine aminotransferase (ALT) normalization within 30 days of diagnosis. All patients received IV methylprednisolone as an initial rejection treatment. Immunofluorescence was performed on archived formalin-fixed paraffin embedded (FFPE) liver biopsy tissue. Liver biopsies from 60 episodes of late ACR in 54 patients were included in the analysis, of which 33 were DR (55%). Anti-thymocyte globulin was only required in the DR group. The frequency of liver-infiltrating CD20+ and CD8+ lymphocytes and the prevalence of autoantibodies were higher in the DR group. In univariate logistic regression analysis, serum gamma-glutamyl transpeptidase (GGT) level at diagnosis, but not ALT, Banff score or presence of DSA, predicted DR. Higher serum GGT level, presence of autoantibodies, and increased CD8+ T-cell infiltration portends DR in late ACR treatment in children.

Multi-Parameter Analysis of Disseminated Tumor Cells (DTCs) in Early Breast Cancer Patients with Hormone-Receptor-Positive Tumors.

Patients with hormone-receptor-positive (HR+) breast cancer are at increased risk for late recurrence. One reason might be disseminated tumor cells (DTCs), which split off in the early stages of the disease and metastasize into the bone marrow (BM). We developed a novel multi-parameter immunofluorescence staining protocol using releasable and bleachable antibody-fluorochrome-conjugates. This sequential procedure enabled us to analyze six distinct phenotypical and therapy-related markers on the same DTC. We characterized BM aspirates from 29 patients with a HR+ tumor and a known positive DTC status-based on the standardized detection of epithelial cells in BM. Using the immunofluorescence staining, a total of 153 DTCs were detected. Luminal A patients revealed a higher DTC count compared with luminal B. The majority of the detected DTCs were CK-positive (128/153). However, in 16 of 17 luminal A patients we found HER2-positive DTCs. We detected CK-negative DTCs (25/153) in 12 of 29 patients. Of those cells, 76% were Ki67-positive and 68% were HER2-positive. Moreover, we detected DTC clusters consisting of mixed characteristics in 6 of 29 patients. Using sequential multi-parameter imaging made it possible to identify distinct DTC profiles not solely based on epithelial features. Our findings indicate that characterization rather than quantification of DTCs might be relevant for treatment decisions.

Checkpoint Blockade-Induced Dermatitis and Colitis Are Dominated by Tissue-Resident Memory T Cells and Th1/Tc1 Cytokines.

Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.

Abstract 1952: Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization

Abstract Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and stratification of cancer patients. AccuCyte® is a density-based unbiased isolation method that transfers nucleated cells from whole blood to slides for the characterization of CTCs and other rare cells. RarePlex® panel kits are IF staining reagents used on automated slide staining instruments to label cells to differentiate CTCs from white blood cells (WBC). CyteFinder® is a seven-channel automated fluorescent imaging system that rapidly scans microscope slides and applies machine learning algorithms to identify CTCs. Together, these technologies provide an end-to-end solution for CTC characterization. For analysis, blood is drawn into AccuCyte blood collection tubes (BCTs) containing a preservative which maintains cell properties prior to processing onto slides. Once slides are prepared, they can be stored at -20°C without significant biomarker degradation. This flexible workflow allows investigators to bank samples for batch analysis and to begin sample collection prior to validating the IF assay to be used. This study was designed to evaluate: (1) stability time between collection in the AccuCyte BCT and sample processing; (2) performance of an improved version of the AccuCyte kit with higher nucleated cell isolation capacity; and (3) storage time that AccuCyte prepared slides can be banked frozen prior to staining. The study was performed using model CTCs and cancer patient samples. Metrics to determine performance were CTC recovery and mean fluorescence intensity (MFI) of biomarker expression. Our results demonstrate that the AccuCyte BCT preserves blood components for at least 5 days after collection without significant effect on CTC recovery or biomarker expression. The latest version of the AccuCyte kit demonstrated a higher cell isolation capacity and could collect up to 60% more nucleated blood cells than the previous version, increasing CTC recovery. The increased capacity was demonstrated in patients treated with hematopoietic growth factors, whose WBC count was significantly higher than the normal range. Finally, accelerated-aging study results demonstrated that AccuCyte-prepared slides can be stored at -20°C for at least 4 years without significant effect on most biomarkers tested. In conclusion, enhancements to the AccuCyte-CyteFinder platform reported here increase flexibility and performance for analysis of CTCs in global clinical trials by allowing longer periods of time before collected blood samples need to be processed and by extending the length of time processed slides can be banked before they are stained. Citation Format: Arturo B. Ramirez, Lillian Costandy, Brady S. Gardner, Ryan H. Huston, A Anders Larson Tevis, Casey E. Helmicki, Alisa C. Clein, Daniel E. Sabath, Joshua J. Nordberg, Tad C. George. Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1952.

Breaking the Immune Complexity of the Tumor Microenvironment Using Single-Cell Technologies.

Tumors are not a simple aggregate of transformed cells but rather a complicated ecosystem containing various components, including infiltrating immune cells, tumor-related stromal cells, endothelial cells, soluble factors, and extracellular matrix proteins. Profiling the immune contexture of this intricate framework is now mandatory to develop more effective cancer therapies and precise immunotherapeutic approaches by identifying exact targets or predictive biomarkers, respectively. Conventional technologies are limited in reaching this goal because they lack high resolution. Recent developments in single-cell technologies, such as single-cell RNA transcriptomics, mass cytometry, and multiparameter immunofluorescence, have revolutionized the cancer immunology field, capturing the heterogeneity of tumor-infiltrating immune cells and the dynamic complexity of tenets that regulate cell networks in the tumor microenvironment. In this review, we describe some of the current single-cell technologies and computational techniques applied for immune-profiling the cancer landscape and discuss future directions of how integrating multi-omics data can guide a new “precision oncology” advancement.

388 A trial to evaluate the safety, immunogenicity, and clinical activity of a helper peptide vaccine plus PD-1 blockade (Mel64, PATHVACS: PD-1 antibody and T-helper vaccine and correlative studies)

BackgroundA multipeptide vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe and immunogenic and has clinical activity. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus PD1 blockade.MethodsParticipants with measurable advanced melanoma, age ≥ 18 years, and ECOG performance status 0–1 were administered 6MHP vaccine intradermally and subcutaneously in an incomplete Freund’s adjuvant on days (D) 1, 8, 15, 43, 64, and 85. Pembrolizumab 200 mg was administered intravenously every 3 weeks for up to two years. Biopsies of accessible tumors at baseline and D22 were analyzed by multiparameter immunofluorescence histology. Primary endpoints were safety (CTCAE 4.03) and immunogenicity (ex vivo IFNγ ELIspot assay). Secondary and exploratory endpoints included changes in the tumor microenvironment (TME), and clinical outcomes.ResultsTwenty-two eligible participants were enrolled and treated, including 6 naïve to PD-1 Ab and 16 anti-PD-1 Ab-experienced. Median follow-up was 20 months. Treatment-related adverse events (any grade) experienced by &gt;20% were injection site reactions, fatigue, anemia, nausea, fever, bruising, and rash. Treatment-related dose limiting toxicities (grade 3 elevated AST, skin ulcer, or uveitis) were observed in 3 (14%), which did not cross the study safety bound. Objective clinical responses were observed in 23% (1 CR, 4 PR), including 4/6 anti-PD-1 Ab-naive (67%) and one 1/16 anti-PD1 Ab-experienced (6%). Four participants (18%) had SD as best radiographic response (18%), all in the Ab-experienced cohort. T cell responses to 6MHP were detected in seven participants (32%) by week 13 and were associated with clinical response (CR/PR 80% vs. SD/PD 18%; p = 0.01). Overall survival was prolonged for anti-PD-1 Ab naïve vs experienced (p = 0.0048), for those with T cell response (p = 0.045, landmark analysis after week 13), and for those with objective response (p = 0.0148). TME evaluation in 12 participants revealed significant increases by D22 in the densities (per mm2) of CD8+ T cells (p = 0.0186), CD20+ B cells (P = 0.002), and Tbet+ cells (p = 0.034).ConclusionsIn patients with advanced melanoma, combined treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral T and B cells, as well as Th1 (Tbet+) cells, and induced T cell responses that were associated with objective response and with overall survival. The promising objective response rate and overall survival in patients naive to PD1 blockade supports consideration of a larger study to assess definitive benefit in that clinical setting.AcknowledgementsWe acknowledge the support of Merck for providing pembrolizumab at no charge, and the University of Virginia Cancer Center Support grant P30 CA044579 for support of shared resource facilities.Trial RegistrationThe clinical trial Mel64 (PATHVACS) is registered with Clinicaltrials.gov (NCT02515227).Ethics ApprovalThe clinical trial Mel64 (PATHVACS) was performed with IRB (#18174) and FDA approval (IND #10825) and is registered with Clinicaltrials.gov (NCT02515227). Written informed consent was obtained from each participant prior to participation in the study.

Coronavirus Disease 2019 (COVID-19) Coronary Vascular Thrombosis: Correlation with Neutrophil but Not Endothelial Activation

Severe coronavirus disease 2019 (COVID-19) increases the risk of myocardial injury that contributes to mortality. This study used multiparameter immunofluorescence to extensively examine heart autopsy tissue of 7 patients who died of COVID-19 compared to 12 control specimens, with or without cardiovascular disease. Consistent with prior reports, no evidence of viral infection or lymphocytic infiltration indicative of myocarditis was found. However, frequent and extensive thrombosis was observed in large and small vessels in the hearts of the COVID-19 cohort, findings that were infrequent in controls. The endothelial lining of thrombosed vessels typically lacked evidence of cytokine-mediated endothelial activation, assessed as nuclear expression of transcription factors p65 (RelA), pSTAT1, or pSTAT3, or evidence of inflammatory activation assessed by expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor, or von Willebrand factor (VWF). Intimal EC lining was also generally preserved with little evidence of cell death or desquamation. In contrast, there were frequent markers of neutrophil activation within myocardial thrombi in patients with COVID-19, including neutrophil-platelet aggregates, neutrophil-rich clusters within macrothrombi, and evidence of neutrophil extracellular trap (NET) formation. These findings point to alterations in circulating neutrophils rather than in the endothelium as contributors to the increased thrombotic diathesis in the hearts of COVID-19 patients.

LBA02-05 NAXIVA - A PHASE II NEOADJUVANT STUDY OF AXITINIB FOR REDUCING EXTENT OF VENOUS TUMOR THROMBUS IN CLEAR CELL RENAL CELL CANCER WITH VENOUS INVASION: TRANSLATIONAL RESULTS

You have accessJournal of UrologyLate-breaking Abstract II - Malignant1 Sep 2021LBA02-05 NAXIVA - A PHASE II NEOADJUVANT STUDY OF AXITINIB FOR REDUCING EXTENT OF VENOUS TUMOR THROMBUS IN CLEAR CELL RENAL CELL CANCER WITH VENOUS INVASION: TRANSLATIONAL RESULTS Sarah J Welsh, James Jones, Stephan Ursprung, Christopher Smith, Ferdia Gallagher, and Grant Stewart Sarah J WelshSarah J Welsh More articles by this author , James JonesJames Jones More articles by this author , Stephan UrsprungStephan Ursprung More articles by this author , Christopher SmithChristopher Smith More articles by this author , Ferdia GallagherFerdia Gallagher More articles by this author , and Grant StewartGrant Stewart More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002149.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Venous tumor thrombus (VTT) extension occurs in 4-15% cases of renal cell cancer (RCC). Although surgery is performed with curative intent, mortality is high (5-15%). The NAXIVA trial provided the first level II evidence in this patient group, assessing the response of VTT to axitinib (TKI). The primary endpoint of percentage of evaluable patients with an improvement in VTT according to the Mayo classification was positive at 26.58%. 35.29% had a change in surgical approach to a less invasive option. Median % reduction in VTT height was 21.49%. Response rate (RECIST) was 61.90% SD, 14.29% PR, 9.52% PD. We now present the results of translational studies examining the role of the tumor microenvironment (TME) and response to axitinib. METHODS: NAXIVA was a single arm, single agent, multi-center, phase 2 feasibility study of 8 weeks axitinib in RCC patients with clear cell RCC prior to nephrectomy/thrombectomy. 21 patients were recruited (15/Dec/2017-06/Jan/2020) at 5 UK sites. Extensive translational sampling (tissue, blood, urine) was performed before starting axitinib, during treatment and at nephrectomy. Samples were processed and analysed for ctDNA (shallow whole genome sequencing), TME (multiparameter immunofluorescence, flow cytometry), and cytokines (multiplex assays). RESULTS: Consistent with previous studies showing low detection of ctDNA in RCC, only 3/21 patients had detectable ctDNA at baseline (2 in plasma, 1 in urine); there was no concordance in the levels or composition of ctDNA between the plasma and urine. No patients with detectable ctDNA at baseline showed a RECIST response or had a change in surgical approach to axitinib. Consistent with its mechanism of action, axitinib treatment reduced vessel density (CD34+ and CD31+CD34+) in all patients. There was a trend towards responders showing higher baseline vessel density and area, with a greater reduction after 8 weeks treatment than non-responders. Interestingly, responders also demonstrated lower CD8+PD1+ tumor infiltration consistent with previous data suggesting such patients may respond better to TKIs than immunotherapy. No individual cytokine or peripheral immune cell-based biomarkers have predicted response but multiparameter analysis is on-going. CONCLUSIONS: NAXIVA has provided unique prospective data on neoadjuvant axitinib to down stage IVC VTT and reduce extent of surgery. Translational work has identified potential biomarkers for validation in future studies, with work on-going. Source of Funding: Educational grant from Pfizer © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1177-e1177 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sarah J Welsh More articles by this author James Jones More articles by this author Stephan Ursprung More articles by this author Christopher Smith More articles by this author Ferdia Gallagher More articles by this author Grant Stewart More articles by this author Expand All Advertisement Loading ...

Comparison of combined positive score, Salgado TIL score, immunofluorescence and single cell RNA sequencing for predicting response to therapy

<h3>Objectives:</h3> Immune cell infiltration is associated with positive ovarian cancer prognosis and is used to predict response to emerging immunotherapies. Multiple methods are used both clinically and in research to quantify immune infiltration, but there is a lack of consensus on the best method. The objective of this study was to compare four methods of measuring immune infiltration: immunohistochemistry for PDL1 (Combined Positive Score - CPS), H&E analysis of immune infiltration (Salgado TIL score), multiparameter immunofluorescence (IHC), and single cell RNA sequencing (scRNAseq). This exploratory work compares these four methods and calculates associations with disease outcomes. <h3>Methods:</h3> Thirty biopsies were collected. Sections were stained with anti-PDL1 or H&E and scored by a molecular pathologist to generate CPS and Salgado TIL scores. Additional slides were stained with antibodies for CD3, CD8, CD56, CD19, FOXP3, cytokeratin, and DAPI. Whole slides were analyzed using CD3 and CD8 density to quantify infiltration. Single cell gene expression was measured using the 10x Genomics Single Cell 3' Protocol. Sequencing was performed to a depth of at least 50,000 paired-end reads per cell. Cells were annotated using the Seurat and SingleR R packages. Correlation coefficients of immune cell infiltration estimates by the four methods were generated and visualized on scatter plots. <h3>Results:</h3> In evaluating immune cell infiltration, there was a moderate correlation between Salgado TIL scores and IHC with an R-squared value of 0.59. In evaluating T cell populations, CPS and scRNASeq did not correlate, with R-squared value of 0.0167. In our dataset, a higher CPS score was positively associated with platinum sensitivity. <h3>Conclusions:</h3> Single methodologies to estimate immune infiltration may not be sufficient to capture the complexity of the tumor microenvironment. Even though the correlation between methods was not high, it is possible that each method measures a different aspect of immune cell infiltration and the combination of methods may improve our ability to predict response to immunotherapies. Further analyses will be required to determine the biological properties that result in the different estimates.

Myeloid Cells Are Enriched in Tonsillar Crypts, Providing Insight into the Viral Tropism of Human Papillomavirus

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. Laser-capture 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 HPV-associated tonsil cancers co-express the same immune checkpoints, providing insight into potential novel immunotherapeutic targets for HPV-associated head and neck cancers.

Abstract 600: Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin

Abstract Protein expression of synaptophysin (SYP) is characteristic of neuroendocrine subtype tumors. In prostate cancer, neuroendocrine differentiation is correlated with disease progression, poor prognosis, and treatment resistance. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and patient stratification. Here we describe the validation of a biomarker IF assay for enumeration of CTCs and characterization of their SYP expression. The 0920-VB SYP CTC assay workflow includes processing blood samples to slides (AccuCyte® Sample Preparation System), staining slides with a panel of fluorescent markers (RarePlex® Staining Kit and Ventana® DISCOVERY® ULTRA immunostaining system), and multiparameter imaging and analysis (CyteFinder® Instrument). The panel consists of a nuclear dye, and antibodies against cytokeratins and EpCAM (to identify epithelial CTCs), CD45 (to exclude white blood cells) and SYP. Both clinical and spike-in samples were used for assay validation. The model CTCs used to generate spike-in samples included 22Rv1 (prostate carcinoma, SYP positive) and BT-474 (breast carcinoma, SYP negative). Performance metrics for the assay included accuracy, sensitivity, specificity, repeatability, and intermediate precision of SYP detection and CTC enumeration. Single-cell SYP mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. An MFI threshold for SYP positivity was established by maximizing the classification accuracy of the positive and negative cell lines. Using this threshold, 98.7% of 22Rv1 cells were correctly classified as SYP-positive, and 99.7% of BT474 cells were correctly classified as SYP-negative with an inter-run coefficient of variation of 13.9% for the 22Rv1 cell line. In clinical prostate, breast and colon cancer patient samples, subsets of CTCs were found to be SYP positive with the expected cytoplasmic localization of the marker. In summary, this liquid biopsy assay provides an analytically sensitive and specific method for CTC enumeration and SYP biomarker expression analysis, allowing non-invasive detection of neuroendocrine differentiation. Citation Format: Jennifer Chow, A Anders Tevis, Edward Lo, Alisa Clein, Daniel E. Sabath, Arturo B. Ramirez, Eric P. Kaldjian, Tad George. Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 600.

Talimogene laherparepvec upregulates immune-cell populations in non-injected lesions: findings from a phase II, multicenter, open-label study in patients with stage IIIB–IVM1c melanoma

BackgroundTalimogene laherparepvec (T-VEC), an oncolytic virus, was designed to selectively replicate in and lyse tumor cells, releasing tumor-derived antigen to stimulate a tumor-specific immune response.MethodsIn this phase II study in...

Abstract 5368: A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer

Abstract Lung cancer biopsy can be difficult to perform successfully; it has a 30% adverse event risk and often provides insufficient material to test. Therefore, a liquid biopsy method to analyze the tumor is advantageous. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression. HER2 is a well-known therapeutic target in breast cancer and studies have shown that HER2 status on CTCs may provide prognostic information about response to anti-HER2 therapies. Less is understood about its frequency and clinical importance in non-small cell lung cancer (NSCLC). The RareCyte platform is uniquely suited for CTC identification and phenotypic characterization with a sensitive, accurate, simple and repeatable workflow from blood collection to CTC characterization and single-cell isolation. We developed an assay for detecting HER2 expression on lung CTCs using the RareCyte platform and validated the assay on model CTC spike-in samples with high (BT-474), medium (MDA-MB-453), low (H1650 and OVCAR-3) and absent (MDA-MB-468) HER2 protein levels. Blood was processed to slides using the AccuCyte Sample Preparation System and stained with anti-HER2 together with the RarePlex Kit for CTC identification (nuclear dye, anti-CD45 to exclude WBC, and anti-cytokeratin/EpCAM for CTCs). The spike-in samples showed the expected trend in per-cell HER2 mean fluorescence intensity values. The percentage of HER2-positive cells was &amp;gt; 90% for the HER2-high, medium and low cell lines, and &amp;lt; 5% for the HER2-negative cell line. The assay was applied to 10 advanced stage (III or IV) post-treatment NSCLC patient samples. At least 1 CTC was found in 6 samples (range 0-2043) and HER2 expression was confirmed in 5 out of the 6 samples, indicating this assay is useful for characterizing HER2 expression in CTCs in NSCLC. Citation Format: Edward Lo, Daniel Campton, Lillian Costandy, Heather Itamoto, Ryan Huston, Braden Gardner, Steve Reese, Arturo Ramirez, Eric P. Kaldjian, Tad George, I-Ming Wang, Steven Pirie-Shepherd. A multiparameter assay for HER2 protein detection on circulating tumor cells in non-small cell lung cancer [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 5368.

Unraveling the Complexity of the Cancer Microenvironment With Multidimensional Genomic and Cytometric Technologies.

Cancers are characterized by extensive heterogeneity that occurs intratumorally, between lesions, and across patients. To study cancer as a complex biological system, multidimensional analyses of the tumor microenvironment are paramount. Single-cell technologies such as flow cytometry, mass cytometry, or single-cell RNA-sequencing have revolutionized our ability to characterize individual cells in great detail and, with that, shed light on the complexity of cancer microenvironments. However, a key limitation of these single-cell technologies is the lack of information on spatial context and multicellular interactions. Investigating spatial contexts of cells requires the incorporation of tissue-based techniques such as multiparameter immunofluorescence, imaging mass cytometry, or in situ detection of transcripts. In this Review, we describe the rise of multidimensional single-cell technologies and provide an overview of their strengths and weaknesses. In addition, we discuss the integration of transcriptomic, genomic, epigenomic, proteomic, and spatially-resolved data in the context of human cancers. Lastly, we will deliberate on how the integration of multi-omics data will help to shed light on the complex role of cell types present within the human tumor microenvironment, and how such system-wide approaches may pave the way toward more effective therapies for the treatment of cancer.

Association of a Type 2-Polarized T Cell Phenotype With Methotrexate Nonresponse in Patients With Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated through complex immunologic pathways. Among RA patients receiving low-dose methotrexate (MTX) monotherapy, approximately one-half exhibit a meaningful clinical response within the first 6 months of starting treatment. Whether baseline immune phenotypes differ between subsequent MTX responders and nonresponders is unknown. This study utilized comprehensive T cell immunophenotyping to identify specific immunologic pathways associated with MTX-nonresponsive joint inflammation in patients with RA. In total, 32 patients with recent-onset RA were treated with MTX therapy. After 6 months, 15 patients were categorized as responders and 17 as nonresponders. Comprehensive blood T cell immunophenotyping, using multiparameter immunofluorescence flow cytometry analyses, was performed at baseline and following 6 months of treatment. Baseline measures of disease activity (Disease Activity Score in 28 joints [DAS28], C-reactive protein level, and erythrocyte sedimentation rate) did not differ between MTX responders and nonresponders following MTX treatment. Frequencies of CD4+ and CD8+ T cells were skewed to favor higher CD4:CD8 T cell ratios in MTX responders compared to nonresponders (P < 0.05). The proportion of inducible costimulator-expressing Treg cells was significantly greater among MTX nonresponders. Interleukin-13 (IL-13)-producing, but not interferon-γ- or IL-17-producing, CD4+ effector memory T (Tem) cells were significantly more frequent in MTX nonresponders (P < 0.05). The ratio of IL-13+:IL-17+ Tem cells among CD4+ Tem cells was 1.9-fold higher in MTX nonresponders compared to responders (P < 0.05). Both the CD4:CD8 T cell ratio and the frequency of IL-13+CD4+ Tem cells correlated with changes in the DAS28 score following MTX treatment, whereas T cell expression of immune checkpoint inhibitor markers (CTLA-4, programmed death 1, and T cell immunoglobulin and mucin domain-containing protein 3) did not differ between MTX responders and nonresponders. We observed a bias toward type 2-polarized T cell inflammatory responses in the peripheral blood of MTX-nonresponsive RA patients. Targeting the IL-13+CD4+ T cell pathway could be a new therapeutic strategy in RA patients whose disease remains resistant to MTX.

A subset of mature neutrophils contains the strongest PMN-MDSC activity in blood and tissue of patients with head and neck cancer

Abstract A high neutrophil-to-lymphocyte ratio in the circulation and high frequencies of tumor-associated neutrophils (TAN) in malignant tissue are associated with poor outcome and tumor progression in patients with cancer. It is hypothesized that immunosuppressive neutrophil activity (aka PMN-MDSC activity) contributes to this effect. However, the exact cellular identity of human PMN-MDSC is still under debate. In this study, we sought to identify the neutrophil subset that contained the highest T cell suppressive activity. To this end, we purified different subsets of circulating neutrophils by FACS and performed multi-parameter immunofluorescence together with digital pathology on 2-D and 3-D tumor tissue samples. We found that a population of circulating, mature, arginase I+ neutrophils that co-purified with mononuclear cells in density gradients, most potently suppressed T cell function in multiple in vitro assays. These PMN-MDSC were also superior to monocytic MDSC in T cell suppression. Using a novel technology of tissue whole mount labelling, clearing and imaging we derived 3-D spatial maps of neutrophil – T cell interaction in human tumors. We found that T cells, which were conjugated to arginase I+, myeloperoxidase+ TAN, had significantly reduced expression of proliferation and cytotoxicity markers. In patients, frequent conjugation of T cells to those PMN-MDSC was associated with poor prognosis. In contrast to circulating PMN-MDSC, tissue PMN-MDSC expressed high amounts of LOX-1 (oxidized low density lipoprotein receptor 1) and a high intratumoral frequency of LOX-1+ PMN-MDSC was associated with poor survival. We identified and characterized PMN-MDSC activity in human cancer patients.

Abstract P4-06-09: Pick-Seq®: Multi-parameter imaging combined with RNA sequencing of tissue section micro-regions for spatial analysis in breast carcinoma

Abstract Multi-parameter tissue imaging allows for a contextual understanding of tumor cells in relation to the tumor microenvironment. Pick-Seq is a novel workflow uniquely enabled by the RareCyte CyteFinder® Instrument that combines interrogation of multiple protein markers with investigation of gene expression from selected micro-regions on tissue slides. In this study, formalin-fixed, paraffin-embedded tonsil sections and OCT-embedded, frozen breast cancer sections were stained with epithelial and lymphocyte markers by multi-parameter immunofluorescence (IF) and scanned with the CyteFinder Instrument to identify regions of interest. Based on IF staining patterns, 40 μm diameter micro-regions were retrieved with the integrated CytePicker® Retrieval Module. Micro-region RNA was isolated for whole transcriptome amplification, library preparation, and sequencing. Resulting data were analyzed for differential gene expression, cell composition, and T cell receptor (TCR) sequences. Transcriptomic analysis of tonsil micro-regions confirmed increased expression of B cell markers in follicles and T cell markers in the T cell zone. CIBERSORT revealed distinct cellular compositions between T cell zones and the B cell follicles. Principle component analysis of gene expression found that micro-regions retrieved from the two follicles clustered independently from each other, and from the T cell zone micro-regions. Fragments per kilobase million (FPKM) analysis revealed differing expression of genes, particularly CD19 and CD21, between the adjacent follicles. Subsequent staining confirmed differential protein expression, indicating that only one follicle contained a germinal center. In breast carcinoma, regions were identified for CytePicker retrieval that included (1) tumor cells, (2) tumor cells with interspersed tumor infiltrating lymphocytes (TIL), or (3) adjacent lymphoid aggregates. CIBERSORT analysis demonstrated the presence of T cell-associated transcriptomic profiles in lymphoid aggregates and in TIL-containing micro-regions that were proportional to the number of T cells retrieved. Aligned RNA-seq reads were further analyzed to identify TCR α and β chain sequences from retrieved TILs. These data establish the potential of combining multi-parameter IF microscopy with deep RNA sequencing as a powerful tool for investigation and biomarker discovery in the immuno-oncology of breast cancer. Citation Format: Nolan Ericson, Rebecca Podyminogin, Jennifer Chow, Jia-Ren Lin, Yu-An Chen, Zoltan Maliga, Peter Sorger, Kyla Teplitz, Eric Kaldjian, Tad George. Pick-Seq®: Multi-parameter imaging combined with RNA sequencing of tissue section micro-regions for spatial analysis in breast carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-09.