Porphyromonas gingivalis, a bridge between oral health and immune evasion in gastric cancer.

Porphyromonas gingivalis (P. gingivalis) is a gram-negative oral pathogen associated with chronic periodontitis. Previous studies have linked poor oral health and periodontitis with oral cancer. Severe cases of periodontal disease can result in advanced periodontitis, leading to tissue degradation, tooth loss, and may also correlate with higher gastric cancer (GC) risk. In fact, tooth loss is associated with an elevated risk of cancer. However, the clinical evidence for this association remains inconclusive. Periodontitis is also characterized by chronic inflammation and upregulation of members of the Programmed Death 1/PD1 Ligand 1 (PD1/PDL1) axis that leads to an immunosuppressive state. Given that chronic inflammation and immunosuppression are conditions that facilitate cancer progression and carcinogenesis, we hypothesize that oral P. gingivalis and/or its virulence factors serve as a mechanistic link between oral health and gastric carcinogenesis/GC progression. We also discuss the potential impact of P. gingivalis' virulence factors (gingipains, lipopolysaccharide (LPS), and fimbriae) on inflammation and the response to immune checkpoint inhibitors in GC which are part of the current standard of care for advanced stage patients.

Historical and Molecular Perspectives on the Presence of Helicobacter pylori in Latin America: A Niche to Improve Gastric Cancer Risk Assessment.

Helicobacter pylori (H. pylori) is responsible for causing chronic gastritis, which can cause peptic ulcer and premalignant lesions such as atrophic gastritis, intestinal metaplasia, and dysplasia, with the risk of developing gastric cancer. Recent data describe that H. pylori colonizes the gastric mucosa of more than 50% of the world's population; however, this bacterium has been described as infecting the human population since its prehistory. This review focuses on the populations and subpopulations of H. pylori, differentiated by the polymorphisms present in their constitutive and virulence genes. These genes have spread and associated with different human populations, showing variability depending on their geographical distribution, and have evolved together with the human being. The predominant genotypes worldwide, Latin America and Chile, are described to understand the genetic diversity and pathogenicity of H. pylori in different populations and geographic regions. The high similarity in the sequence of virulence genes between H. pylori strains present in Peruvian and Spanish natives in Latin America suggests a European influence. The presence of cagA-positive strains and vacA s1 m1 allelic variants is observed with greater prevalence in Chilean patients with more severe gastrointestinal diseases and is associated with its geographical distribution. These findings highlight the importance of understanding the genetic diversity of H. pylori in different regions of the world for a more accurate assessment of the risk of associated diseases and their potential impact on health.

Minireview: functional roles of tissue kallikrein, kinins, and kallikrein-related peptidases in lung cancer.

Despite campaigns and improvements in detection and treatment, lung cancer continues to increase worldwide and represents a major public health problem. One approach to treating patients suffering from lung cancer is to target surface receptors overexpressed on tumor cells, such as GPCR-family kinin receptors, and proteases that control tumor progression, such as kallikrein-related peptidases (KLKs). These proteases have been visualized in recent years due to their contribution to the progression of cancers, such as prostate and ovarian cancer, facilitating the invasive and metastatic capacity of tumor cells in these tissues. In fact, KLK3 is the specific prostate antigen, the only tissue-specific biomarker used to diagnose this malignancy. In lung cancer to date, evidence indicates that KLK5, KLK6, KLK8, KLK11, and KLK14 are the major peptidases regulated and involved in its progression. The expression levels of KLKs in this neoplasm are modulated by the secretome of the different cell types present in the tumor microenvironment, the cancer subtype and the tumor stage, among others. Considering the multiple functions of kinin receptors and KLKs, this review highlights their roles, even considering the SARS-CoV-2 effects. Since lung cancer is often diagnosed in advanced stages, our efforts should focus on early diagnosis, validating for example specific KLKs, especially in high-risk populations such as smokers and people exposed to carcinogenic fumes, oil fields, and contaminated workplaces, unexplored fields to investigate. Furthermore, their modulation could be considered as a promising approach in lung cancer therapeutics.

Intersections between innate immune response and gastric cancer development.

Worldwide, gastric cancer (GC) is the fifth most commonly diagnosed malignancy. It has a reduced prevalence but has maintained its poor prognosis being the fourth leading cause of deaths related to cancer. The highest mortality rates occur in Asian and Latin American countries, where cases are usually diagnosed at advanced stages. Overall, GC is viewed as the consequence of a multifactorial process, involving the virulence of the Helicobacter pylori (H. pylori) strains, as well as some environmental factors, dietary habits, and host intrinsic factors. The tumor microenvironment in GC appears to be chronically inflamed which promotes tumor progression and reduces the therapeutic opportunities. It has been suggested that inflammation assessment needs to be measured qualitatively and quantitatively, considering cell-infiltration types, availability of receptors to detect damage and pathogens, and presence or absence of aggressive H. pylori strains. Gastrointestinal epithelial cells express several Toll-like receptors and determine the first defensive line against pathogens, and have been also described as mediators of tumorigenesis. However, other molecules, such as cytokines related to inflammation and innate immunity, including immune checkpoint molecules, interferon-gamma pathway and NETosis have been associated with an increased risk of GC. Therefore, this review will explore innate immune activation in the context of premalignant lesions of the gastric epithelium and established gastric tumors.

Recombinant Domain of Flagellin Promotes In Vitro a Chemotactic Inflammatory Profile in Human Immune Cells Independently of a Dendritic Cell Phenotype.

Flagellin is the major component of the flagellum in gram-positive and -negative bacteria and is also the ligand for the Toll-like receptor 5 (TLR5). The activation of TLR5 promotes the expression of proinflammatory cytokines and chemokines and the subsequent activation of T cells. This study evaluated a recombinant domain from the amino-terminus D1 domain (rND1) of flagellin from Vibrio anguillarum, a fish pathogen, as an immunomodulator in human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs). We demonstrated that rND1 induced an upregulation of proinflammatory cytokines in PBMCs, characterized at the transcriptional level by an expression peak of 220-fold for IL-1ß, 20-fold for IL-8, and 65-fold for TNF-α. In addition, at the protein level, 29 cytokines and chemokines were evaluated in the supernatant and were correlated with a chemotactic signature. MoDCs treated with rND1 showed low levels of co-stimulatory and HLA-DR molecules and kept an immature phenotype with a decreased phagocytosis of dextran. We probed that rND1 from a non-human pathogen promotes modulation in human cells, and it may be considered for further studies in adjuvant therapies based on pathogen-associated patterns (PAMPs).

Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes.

The main objective of this study was to estimate the performance, under local epidemiological conditions, of two in-house ELISA assays for the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulins. A total of 94 serum samples were used for the assessment, where 44 corresponded to sera collected before the pandemic (free of SARS-CoV-2 antibodies), and 50 sera were collected from confirmed COVID-19 patients admitted to the main public hospital in the city of Valdivia, southern Chile. The Nucleocapsid (Np) and the receptor-binding domain (RBD) proteins were separately used as antigens (Np and RBD ELISA, respectively) to assess their diagnostic performance. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off that maximized the sensitivity (Se) and specificity (Sp) of the ELISA assays. Np ELISA had a mean Se of 94% (95% CI = 83.5-98.8%) and a mean Sp of 100% (95% CI = 92.0-100%), with an OD 450 nm positive cut-off value of 0.88. On the other hand, RBD ELISA presented a mean Se of 96% (95% CI = 86.3-99.5%) and a mean Sp of 90% (95% CI = 78.3-97.5%), with an OD 450 nm positive cut off value of 0.996. Non-significant differences were observed between the Se distributions of Np and RBD ELISAs, but the latter presented a significant lower Sp than Np ELISA. In parallel, collected sera were also analyzed using a commercial lateral flow chromatographic immunoassay (LFCI), to compare the performance of the in-house ELISA assays against a commercial test. The LFCI had a mean sensitivity of 94% (95% CI = 87.4-100%) and a mean specificity of 100% (95% CI = 100-100%). When compared to Np ELISA, non-significant differences were observed on the performance distributions. Conversely, RBD ELISA had a significant lower Sp than the LFCI. Although, Np ELISA presented a similar performance to the commercial test, this was 2.5 times cheaper than the LFCI assay (labor cost not considered). Thus, the in-house Np ELISA could be a suitable alternative tool, in resource limited environments, for the surveillance of SARS-CoV-2 infection, supporting further epidemiological studies.

The Armed Conflict and the Impact on Patients With Cancer in Ukraine: Urgent Considerations.

On February 24, 2022, a war began within the Ukrainian borders. At least 3.0 million Ukrainian inhabitants have already fled the country. Critical infrastructure, including hospitals, has been damaged. Children with cancer were urgently transported to foreign countries, in an effort to minimize interruption of their life-saving treatments. Most adults did not have that option. War breeds cancer-delaying diagnosis, preventing treatment, and increasing risk. We project that a modest delay in care of only 4 months for five prevalent types of cancer will lead to an excess of over 3,600 cancer deaths in the subsequent years. It is critical that we establish plans to mitigate that risk as soon as possible.

Quantitative tissue analysis and role of myeloid cells in non-small cell lung cancer.

BACKGROUND: Despite the prominent role of innate immunity in the antitumor response, little is known about the myeloid composition of human non-small cell lung cancer (NSCLC) with respect to histology and molecular subtype. We used multiplexed quantitative immunofluorescence (QIF) to measure the distribution and clinical significance of major myeloid cell subsets in large retrospective NSCLC collections. METHODS: We established a QIF panel to map major myeloid cell subsets in fixed human NSCLC including 4',6-Diamidino-2-Phenylindole for all cells, pancytokeratin for tumor-epithelial cells, CD68 for M1-like macrophages; and CD11b plus HLA-DR to interrogate mature and immature myeloid cell populations such as myeloid derived suppressor cells (MDSCs). We interrogated 793 NSCLCs represented in four tissue microarray-based cohorts: #1 (Yale, n=379) and #2 (Greece, n=230) with diverse NSCLC subtypes; #3 (Yale, n=138) with molecularly annotated lung adenocarcinomas (ADC); and #4 (Yale, n=46) with patient-matched NSCLC and morphologically-normal lung tissue. We examined associations between marker levels, myeloid cell profiles, clinicopathologic/molecular variables and survival. RESULTS: The levels of CD68+ M1 like macrophages were significantly lower and the fraction of CD11b+/HLA-DR- MDSC-like cells was prominently higher in tumor than in matched non-tumor lung tissues. HLA-DR was consistently higher in myeloid cells from tumors with elevated CD68 expression. Stromal CD11b was significantly higher in squamous cell carcinomas (SCC) than in ADC across the cohorts and EGFR-mutated lung ADCs displayed lower CD11b levels than KRAS-mutant tumors. Increased stromal CD68- and HLA-DR-expressing cells was associated with better survival in ADCs from two independent NSCLC cohorts. In SCC, increased stromal CD11b or HLA-DR expression was associated with a trend towards shorter 5-year survival. CONCLUSIONS: NSCLCs display an unfavorable myeloid immune contexture relative to non-tumor lung and exhibit distinct myeloid-cell profiles across histologies and presence of major oncogenic driver-mutations. Elevated M1-like stromal proinflammatory myeloid cells are prognostic in lung ADC, but not in SCC.

Tumor cell SYK expression modulates the tumor immune microenvironment composition in human cancer via TNF-α dependent signaling.

BACKGROUND: The expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK in modulating local adaptive antitumor immune responses remains uncertain. Using detailed analysis of primary human tumors and in vitro models, we reveal the immunomodulatory effect of SYK protein in human solid cancer. METHODS: We spatially mapped SYK kinase in tumor cells, stromal cells and tumor-infiltrating leukocytes (TILs) in 808 primary non-small cell lung carcinomas (NSCLCs) from two cohorts and in 374 breast carcinomas (BCs) from two independent cohorts. We established the associations of localized SYK with clinicopathologic variables and outcomes. The immunomodulatory role of SYK on tumor cells was assessed using in vitro cytokine stimulation, transcriptomic analysis and selective SYK blockade using a small molecule inhibitor. Functional responses were assessed using cocultures of tumor cells with peripheral blood lymphocytes. T cell responses in baseline and post-treatment biopsies from patients with BC treated with a SYK inhibitor in a phase I clinical trial were also studied. RESULTS: Elevated tumor cell or leukocyte SYK expression was associated with high CD4+ and CD8+ TILs and better outcome in both NSCLC and BC. Tumor cell SYK was associated with oncogenic driver mutations in EGFR or KRAS in lung adenocarcinomas and with triple negative phenotype in BC. In cultured tumor cells, SYK was upregulated by TNFα and required for the TNFα-induced proinflammatory responses and T cell activation. SYK blockade after nivolumab in a phase I clinical trial including three patients with advanced triple negative BC reduced TILs and T cell proliferation. Our work establishes the proinflammatory function of tumor cell SYK in lung and breast cancer. SYK signaling in cultured tumor cells is required for T cell activation and SYK blockade limits adaptive antitumor immune responses and tumor rejection in patients with cancer. CONCLUSIONS: Together, our results establish the immunomodulatory role of SYK expression in human solid tumors. This information could be used to develop novel biomarkers and/or therapeutic strategies.

Simultaneous and Spatially-Resolved Analysis of T-Lymphocytes, Macrophages and PD-L1 Immune Checkpoint in Rare Cancers.

Penile, vulvar and anal neoplasms show an incidence lower than 0.5% of the population per year and therefore can be considered as rare cancers but with a dramatic impact on quality of life and survival. This work describes the experience of a Chilean cancer center using multiplexed immunofluorescence to study a case series of four penile cancers, two anal cancers and one vulvar cancer and simultaneous detection of CD8, CD68, PD-L1, Cytokeratin and Ki-67 in FFPE samples. Fluorescent image analyses were performed using open sources for automated tissue segmentation and cell phenotyping. Our results showed an objective and reliable counting of objects with a single or combined labeling or within a specific tissue compartment. The variability was below 10%, and the correlation between analytical events was 0.92-0.97. Critical cell phenotypes, such as TILs, PD-L1+ or proliferative tumor cells were detected in a supervised and unsupervised manner with a limit of detection of less than 1% of relative abundance. Finally, the observed diversity and abundance of the different cell phenotypes within the tumor microenvironment for the three studied tumor types confirmed that our methodology is useful and robust to be applicable for many other solid tumors.

Advances in mass cytometry and its applicability to digital pathology in clinical-translational cancer research.

The development and subsequent adaptation of mass cytometry for the histological analysis of tissue sections has allowed the simultaneous spatial characterization of multiple components. This is useful to find the correlation between the genotypic and phenotypic profile of tumor cells and their environment in clinical-translational studies. In this revision, we provide an overview of the most relevant hallmarks in the development, implementation and application of multiplexed imaging in the study of cancer and other conditions. A special focus is placed on studies based on imaging mass cytometry (IMC) and multiplexed ion beam imaging (MIBI). The purpose of this review is to help our readers become familiar with the verification techniques employed on this tool and outline the multiple applications reported in the literature. This review will also provide guidance on the use of IMC or MIBI in any field of biomedical research.

Graph of graphs analysis for multiplexed data with application to imaging mass cytometry.

Imaging Mass Cytometry (IMC) combines laser ablation and mass spectrometry to quantitate metal-conjugated primary antibodies incubated in intact tumor tissue slides. This strategy allows spatially-resolved multiplexing of dozens of simultaneous protein targets with 1µm resolution. Each slide is a spatial assay consisting of high-dimensional multivariate observations (m-dimensional feature space) collected at different spatial positions and capturing data from a single biological sample or even representative spots from multiple samples when using tissue microarrays. Often, each of these spatial assays could be characterized by several regions of interest (ROIs). To extract meaningful information from the multi-dimensional observations recorded at different ROIs across different assays, we propose to analyze such datasets using a two-step graph-based approach. We first construct for each ROI a graph representing the interactions between the m covariates and compute an m dimensional vector characterizing the steady state distribution among features. We then use all these m-dimensional vectors to construct a graph between the ROIs from all assays. This second graph is subjected to a nonlinear dimension reduction analysis, retrieving the intrinsic geometric representation of the ROIs. Such a representation provides the foundation for efficient and accurate organization of the different ROIs that correlates with their phenotypes. Theoretically, we show that when the ROIs have a particular bi-modal distribution, the new representation gives rise to a better distinction between the two modalities compared to the maximum a posteriori (MAP) estimator. We applied our method to predict the sensitivity to PD-1 axis blockers treatment of lung cancer subjects based on IMC data, achieving 97.3% average accuracy on two IMC datasets. This serves as empirical evidence that the graph of graphs approach enables us to integrate multiple ROIs and the intra-relationships between the features at each ROI, giving rise to an informative representation that is strongly associated with the phenotypic state of the entire image.

Early versus deferred anti-SARS-CoV-2 convalescent plasma in patients admitted for COVID-19: A randomized phase II clinical trial.

BACKGROUND: Convalescent plasma (CP), despite limited evidence on its efficacy, is being widely used as a compassionate therapy for hospitalized patients with COVID-19. We aimed to evaluate the efficacy and safety of early CP therapy in COVID-19 progression. METHODS AND FINDINGS: The study was an open-label, single-center randomized clinical trial performed in an academic medical center in Santiago, Chile, from May 10, 2020, to July 18, 2020, with final follow-up until August 17, 2020. The trial included patients hospitalized within the first 7 days of COVID-19 symptom onset, presenting risk factors for illness progression and not on mechanical ventilation. The intervention consisted of immediate CP (early plasma group) versus no CP unless developing prespecified criteria of deterioration (deferred plasma group). Additional standard treatment was allowed in both arms. The primary outcome was a composite of mechanical ventilation, hospitalization for >14 days, or death. The key secondary outcomes included time to respiratory failure, days of mechanical ventilation, hospital length of stay, mortality at 30 days, and SARS-CoV-2 real-time PCR clearance rate. Of 58 randomized patients (mean age, 65.8 years; 50% male), 57 (98.3%) completed the trial. A total of 13 (43.3%) participants from the deferred group received plasma based on clinical aggravation. We failed to find benefit in the primary outcome (32.1% versus 33.3%, odds ratio [OR] 0.95, 95% CI 0.32-2.84, p > 0.999) in the early versus deferred CP group. The in-hospital mortality rate was 17.9% versus 6.7% (OR 3.04, 95% CI 0.54-17.17 p = 0.246), mechanical ventilation 17.9% versus 6.7% (OR 3.04, 95% CI 0.54-17.17, p = 0.246), and prolonged hospitalization 21.4% versus 30.0% (OR 0.64, 95% CI, 0.19-2.10, p = 0.554) in the early versus deferred CP group, respectively. The viral clearance rate on day 3 (26% versus 8%, p = 0.204) and day 7 (38% versus 19%, p = 0.374) did not differ between groups. Two patients experienced serious adverse events within 6 hours after plasma transfusion. The main limitation of this study is the lack of statistical power to detect a smaller but clinically relevant therapeutic effect of CP, as well as not having confirmed neutralizing antibodies in donor before plasma infusion. CONCLUSIONS: In the present study, we failed to find evidence of benefit in mortality, length of hospitalization, or mechanical ventilation requirement by immediate addition of CP therapy in the early stages of COVID-19 compared to its use only in case of patient deterioration. TRIAL REGISTRATION: NCT04375098.

Biomarker Discovery in Patients with Immunotherapy-Treated Melanoma with Imaging Mass Cytometry.

PURPOSE: Imaging mass cytometry (IMC) is among the first tools with the capacity for multiplex analysis of more than 40 targets, which provides a novel approach to biomarker discovery. Here, we used IMC to characterize the tumor microenvironment of patients with metastatic melanoma who received immunotherapy in efforts to find indicative factors of treatment response. In spite of the new power of IMC, the image analysis aspects are still limited by the challenges of cell segmentation. EXPERIMENTAL DESIGN: Here, rather than segment, we performed image analysis using a newly designed version of the AQUA software to measure marker intensity in molecularly defined compartments: tumor cells, stroma, T cells, B cells, and macrophages. IMC data were compared with quantitative immunofluorescence (QIF) and digital spatial profiling. RESULTS: Validation of IMC results for immune markers was confirmed by regression with additional multiplexing methods and outcome assessment. Multivariable analyses by each compartment revealed significant associations of 12 markers for progression-free survival and seven markers for overall survival (OS). The most compelling indicative biomarker, beta2-microglobulin (B2M), was confirmed by correlation with OS by QIF in the discovery cohort and validated in an independent published cohort profiled by mRNA expression. CONCLUSIONS: Using digital image analysis based on pixel colocalization to assess IMC data allowed us to quantitively measure 25 markers simultaneously on formalin-fixed, paraffin-embedded tissue microarray samples. In addition to showing high concordance with other multiplexing technologies, we identified a series of potentially indicative biomarkers for immunotherapy in metastatic melanoma, including B2M.

Full recombinant flagellin B from Vibrio anguillarum (rFLA) and its recombinant D1 domain (rND1) promote a pro-inflammatory state and improve vaccination against P. salmonis in Atlantic salmon (S. salar).

Flagellin is the major component of the flagellum, and a ligand for Toll-like receptor 5. As reported, recombinant flagellin (rFLA) from Vibrio anguillarum and its D1 domain (rND1) are able to promote in vitro an upregulation of pro-inflammatory genes in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) macrophages. This study evaluated the in vitro and in vivo stimulatory/adjuvant effect for rFLA and rND1 during P. salmonis vaccination in Atlantic salmon (Salmo salar). We demonstrated that rFLA and rND1 are molecules able to generate an acute upregulation of pro-inflammatory cytokines (IL-1ß, IL-8, IL-12ß), allowing the expression of genes associated with T-cell activation (IL-2, CD4, CD8ß), and differentiation (IFNγ, IL-4/13, T-bet, Eomes, GATA3), in a differential manner, tissue/time dependent way. Altogether, our results suggest that rFLA and rND1 are valid candidates to be used as an immuno-stimulant or adjuvants with existing vaccines in farmed salmon.

The Reprimo-Like Gene Is an Epigenetic-Mediated Tumor Suppressor and a Candidate Biomarker for the Non-Invasive Detection of Gastric Cancer.

Reprimo-like (RPRML) is an uncharacterized member of the Reprimo gene family. Here, we evaluated the role of RPRML and whether its regulation by DNA methylation is a potential non-invasive biomarker of gastric cancer. RPRML expression was evaluated by immunohistochemistry in 90 patients with gastric cancer and associated with clinicopathologic characteristics and outcomes. The role of RPRML in cancer biology was investigated in vitro, through RPRML ectopic overexpression. Functional experiments included colony formation, soft agar, MTS, and Ki67 immunofluorescence assays. DNA methylation-mediated silencing was evaluated by the 5-azacytidine assay and direct bisulfite sequencing. Non-invasive detection of circulating methylated RPRML DNA was assessed in 25 gastric cancer cases and 25 age- and sex-balanced cancer-free controls by the MethyLight assay. Downregulation of RPRML protein expression was associated with poor overall survival in advanced gastric cancer. RPRML overexpression significantly inhibited clonogenic capacity, anchorage-independent growth, and proliferation in vitro. Circulating methylated RPRML DNA distinguished patients with gastric cancer from controls with an area under the curve of 0.726. The in vitro overexpression results and the poor patient survival associated with lower RPRML levels suggest that RPRML plays a tumor-suppressive role in the stomach. Circulating methylated RPRML DNA may serve as a biomarker for the non-invasive detection of gastric cancer.

Effects of Radiation on the Tumor Microenvironment.

A malignant tumor consists of malignant cells as well as a wide array of normal host tissues including stroma, vasculature, and immune infiltrate. The interaction between cancer and these host tissues is critical as these host tissues play a variety of roles in supporting or resisting disease progression. Radiotherapy (RT) has direct effects on malignant cells, but, also, critically important effects on these other components of the tumor microenvironment (TME). Given the growing role of immune checkpoint inhibitors and other immunotherapy strategies, understanding how RT affects the TME, particularly the immune compartment, is essential to advance RT in this new era of cancer therapy. The interactions between RT and the TME are complex, affecting the innate and adaptive arms of the immune system. RT can induce both proinflammatory effects and immune suppressive effects that can either promote or impede antitumor immunity. It is likely that the initial proinflammatory effects of RT eventually lead to rebound immune-suppression as chronic inflammation sets in. The exact kinetics and nature of how RT changes the TME likely depends on timing, dose, fractionation, site irradiated, and tumor type. With increased understanding of the effects of RT on the TME, in the future it is likely that we will be able to personalize RT by varying the dose, site, and timing of intervention to generate the desired response to partner with immunotherapy strategies.

Multiplexed (18-Plex) Measurement of Signaling Targets and Cytotoxic T Cells in Trastuzumab-Treated Patients using Imaging Mass Cytometry.

PURPOSE: Imaging mass cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T-cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab to discover associations with trastuzumab benefit. EXPERIMENTAL DESIGN: An antibody panel for detection of 18 targets (pan-cytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, ß-catenin, HER3, MET, EGFR, ERK 1-2, MEK 1-2, PTEN, PI3K p110 α, Akt, mTOR, Ki67, and Histone H3) was used with a selection of trastuzumab-treated patients from the Hellenic Cooperative Oncology Group 10/05 trial (n = 180), and identified a case-control series. RESULTS: Patients that recurred after adjuvant treatment with trastuzumab trended toward a decreased fraction of HER2 ECD pixels over threshold compared with cases without recurrence (P = 0.057). After exclusion of the lowest HER2 expressers, 5-year recurrence events were associated with reduced total extracellular domain (ECD)/intracellular domain (ICD) ratio intensity in tumor (P = 0.044). These observations are consistent with our previous work using quantitative immunofluorescence, but represent the proof on identical cell content. We also describe the association of the ECD of HER2 with CD8 T-cell infiltration on the same slide. CONCLUSIONS: The proximity of CD8 cells as a function of the expression of the ECD of HER2 provides further evidence for the role of the immune system in the mechanism of action of trastuzumab.

Neoadjuvant nivolumab modifies the tumor immune microenvironment in resectable glioblastoma.

Glioblastoma is the most common primary central nervous system malignancy and has a poor prognosis. Standard first-line treatment, which includes surgery followed by adjuvant radio-chemotherapy, produces only modest benefits to survival1,2. Here, to explore the feasibility, safety and immunobiological effects of PD-1 blockade in patients undergoing surgery for glioblastoma, we conducted a single-arm phase II clinical trial (NCT02550249) in which we tested a presurgical dose of nivolumab followed by postsurgical nivolumab until disease progression or unacceptable toxicity in 30 patients (27 salvage surgeries for recurrent cases and 3 cases of primary surgery for newly diagnosed patients). Availability of tumor tissue pre- and post-nivolumab dosing and from additional patients who did not receive nivolumab allowed the evaluation of changes in the tumor immune microenvironment using multiple molecular and cellular analyses. Neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell infiltration and augmented TCR clonal diversity among tumor-infiltrating T lymphocytes, supporting a local immunomodulatory effect of treatment. Although no obvious clinical benefit was substantiated following salvage surgery, two of the three patients treated with nivolumab before and after primary surgery remain alive 33 and 28 months later.

CD68, CD163, and matrix metalloproteinase 9 (MMP-9) co-localization in breast tumor microenvironment predicts survival differently in ER-positive and -negative cancers.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in the cancer immune landscape and their potential as treatment targets or modulators of response to treatment are gaining increasing interest. TAMs display high molecular and functional complexity. Therefore their objective assessment as breast cancer biomarkers is critical. The aims of this study were to objectively determine the in situ expression and significance of TAM biomarkers (CD68, CD163, and MMP-9) in breast cancer and to identify subclasses of patients who could benefit from TAM-targeting therapies. METHODS: We measured CD68, CD163, and MMP-9 protein expression in formalin-fixed paraffin-embedded tissues of breast carcinomas represented in tissue microarray format using multiplexed quantitative immunofluorescence (QIF) in two independent Yale cohorts: cohort A-n = 398, estrogen receptor-positive (ER+) and ER- cases-and the triple-negative breast cancer (TNBC)-only cohort B (n = 160). Associations between macrophage markers, ER status, and survival were assessed. Protein expression measured by QIF was compared with mRNA expression data from the METABRIC study. RESULTS: All three macrophage markers were co-expressed, displaying higher expression in ER- cancers. High pan-macrophage marker CD68 correlated with poorer overall survival (OS) only in ER- cases of cohort A (P = 0.02). High expression of CD163 protein in TAMs was associated with improved OS in ER- cases (cohort A, P = 0.03 and TNBC cohort B, P = 0.04, respectively) but not in ER+ cancers. MMP-9 protein was not individually associated with OS. High expression of MMP-9 in the CD68+/CD163+ TAMs was associated with worse OS in ER+ tumors (P <0.001) but not in ER- cancers. In the METABRIC dataset, mRNA levels followed the co-expression pattern observed in QIF but did not always show the same trend regarding OS. CONCLUSIONS: Macrophage activity markers correlate with survival differently in ER+ and ER- cancers. The association between high co-expression and co-localization of MMP-9/CD163/CD68 and poor survival in ER+ cancers suggests that these cancers may be candidates for macrophage-targeted therapies.