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Purpose: Intraductal papillary mucinous neoplasms (IPMN) are bona fide precursors to pancreatic ductal adenocarcinoma (PDAC). While genomic alterations during multistep IPMN progression have been well cataloged, the accompanying changes within the tumor immune microenvironment (TIME) have not been comprehensively studied. Herein, we investigated TIME-related alterations during IPMN progression, using multiplex immunofluorescence (mIF) coupled with high-resolution image analyses. Experimental Design: Two sets of formalin-fixed, paraffin-embedded tissue samples from surgically resected IPMNs were analyzed. The training set of 30 samples consisted of 11 low-grade IPMN (LG-IPMN), 17 high-grade IPMN (HG-IPMN), and 2 IPMN with PDAC, while a validation set of 93 samples comprised of 55 LG-IPMN and 38 HG-IPMN. The training set was analyzed with two panels of immuno-oncology–related biomarkers, while the validation set was analyzed with a subset of markers found significantly altered in the training set. Results: Cell types indicative of enhanced immune surveillance, including cytotoxic and memory T cells, and antigen-experienced T cells and B cells, were all found at higher densities within isolated LG-IPMNs compared with HG-IPMNs. Notably, the TIME of LG-IPMNs that had progressed at the time of surgical resection (progressor LGD) resembled that of the synchronous HG-IPMNs, underscoring that attenuated immune surveillance occurs even in LG-IPMNs destined for progression. Conclusions: Our findings provide a basis for interception of cystic neoplasia to PDAC, through maintenance of sustained immune surveillance using vaccines and other prevention approaches.

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER1% and HER2-negative) group and the ER/PR-low positive (ER 1-9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1

Background: Lung squamous cell carcinoma (SCC) is the second most common type of non-small cell lung cancer. Immunotherapy is a promising treatment for SCC. However, only a small proportion of unselected SCC patients are able to achieve durable clinical benefit. We leveraged the precious specimens from S1400I trial and sought to explore whether the levels and spatial distributions of tumor-associated immune cells (TAICs) in screening samples are associated with PFS, and OS. Methods: We utilized paraffin tumor tissue provided by the SWOG bank from screening on LungMAP. 82/252 eligible SCC sample from patients treated with nivolumab plus ipilimumab (nivo+ipi; n = 35) or with nivolumab alone (nivo; n = 47) were studied. Image analysis tissue immunoprofiling was conducted using 9 markers in 2 multiplex immunofluorescence panels against cytokeratin, CD3, CD8, granzyme B, CD45RO, and FOXP3, PD1, PD-L1, and CD68. Densities of cells phenotypes (cells/mm2) were assessed and dichotomized as being high (> median) or low (≤ median) in tumor, stroma, and total compartments. Distances from malignant cells (MCs) to cell populations were measured and dichotomized as close from MCs (≤ median) and faraway from MCs (> median) and associated with clinical variables. Clinicopathologic features and data on PFS, and OS were retrieved from the CIDC portal as provided by the LungMap team. Non-parametric tests where utilized to assessment associations of cell phenotypes versus continue or categorical variables and log-rank test for survival analysis. Results: Univariate analysis showed that higher densities of memory T cells in total compartment (HR:0.63, CI:0.40-1.00, P=0.041), antigen-experienced T cells in the stroma (HR:0.60, CI:0.37-0.96, P=0.024) and total compartment (HR:0.60, CI:0.38-0.95, P=0.023) and activated cytotoxic T cells in the tumor compartment (HR:0.55, CI:0.35-0.87, P=0.011) were associated with significative better PFS. In the nivo+ipi arm, higher ratios of cytotoxic T cells to regulatory T cells in the stroma were associated with better PFS. The spatial analysis of these TAICs showed that the presence of activated cytotoxic T cells close to the MCs (median, ≤19.27 µm) was associated with better PFS (P=0.037). Conclusions: Our findings suggest that patients who have a detectable immunosuppressive tumor axis (PD-L1/PD1) with adequate activated cytotoxic T cells close to the tumor cells are the ideal patients for immune therapy to be targetable with these types of treatments. Acknowledgments: Scientific and financial support for: U10CA180888, U10CA180819U24CA224285, U24CA224316, PACT, PPP and FNIH. Citation Format: Edwin Roger Parra, Jiexin Zhang, Mary Redman, Rossana Lazcano, Piubelli Mario, Caddie Laberiano Fernandez, Renganayaki K. Krishna, Shanyu Zhang, Hong Chen, Ganiraju Manyam, Radim Moravec, Ethan Cerami, James Lindsay, Joyce Yu, Roshni Biswas, Stephen Van Nostrand, Dzifa Y. Duose, Jianjun Zhang, Roy Herbst, Gheath Al-Atrash, Kasthuri Kannan, Ignacio I. Wistuba, Scott Gettinger, Lyudmila Bazhenova, Jack Lee, Jianhua Zhang, Cara Haymaker. Infiltration and spatial distribution of immune cells are associated clinical benefit from Nivolumab and Ipilimumab for previously treated patients with stage IV squamous cell lung cancer: an immune biomarker analysis of Phase III SWOG LungMAP S1400I trial [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 2580.

Programmed cell death 1 (PD-1) plays an important role in subsiding immune responses, in promoting self-tolerance through suppressing the activity of T-cells, and in promoting differentiation of regulatory T-cells. One of its ligands, programmed cell death ligand 1 (PD-L1) acts as a checkpoint regulator in immune cells and is also expressed in a wide range of cancer types. Anti-PD therapy modulates immune responses at the tumor site, targets tumor-induced immune defects, and repairs ongoing immune responses. Since drugs that target the PD-1/PD-L1 pathways became available as a cancer treatment, there is need for the use of different antibodies to detect the presence of these proteins in tumoral samples by immunohistochemistry or other assays. Because the detection of these antigens in tumor samples is highly clinically informative for guiding treatment decisions, especially to establish the aptness of a patient to receive anti-PD therapy, it is necessary to have a validation process that guaranties that the test results obtained when using antibodies against these proteins are specific, selective, reproducible, and conducive to quantification of antigen abundance in cancer tissue sections. Here we describe an automated immunohistochemistry staining procedure that can be applied for the validation of multiple anti-PD-L1 antibody clones when used for the staining of formalin-fixed, paraffin-embedded lung cancer tissue sections.

Antibody selection and optimization are crucial to guarantee accurate and reproducible results when using such antibodies for applications such as western blot analysis and immunohistochemistry (IHC). This is especially important when selecting good candidate antibodies that will be used for cancer immunotherapy diagnostics and research. In this chapter, we describe a Western Blot technique as support methodology for the selection and validation of Programmed Cell Death Ligand 1 (PD-L1) antibodies that can be subsequently used in immunohistochemistry applications. Western Blot is a sensitive, specific, and widely available protein characterization technique, used for the detection of specific antigens. PD-L1 is a major immune checkpoint protein that mediates antitumor immune suppression and response, which is routinely detected using IHC in formalin-fixed and paraffin-embedded tissues as part of cancer clinical diagnostic workflows. For this reason, it is critical to define and select the best antibody clones and validate them using different techniques in order to have a reliable detection of positive staining when these antibodies are used in IHC.

Patients with Systemic sclerosis (SSc) presents immune dysregulation, vasculopathy, and fibrosis of the skin and various internal organs. Pulmonary fibrosis leads to SSc-associated interstitial lung disease (ILD), which is the main cause of morbidity and mortality in SSc. Recently autoimmunity to type V collagen (Col V) has been characterized in idiopathic pulmonary fibrosis and show promise to be related to the development in SSc. Our aim was to evaluate autoimmunity to Col V α1(V) and α2(V) chains and to the antigenic peptides of these Col V chains in early-SSc sera employing lung tissue of SSc-ILD, as antigen source. We found that sera samples from patients with early-SSc were reactive to Col V (41.18%) and presented immunoreactivity for Col5A1(1.049) and Col5A1(1.439) peptides. The IgG isolated from early-SSc patients-anti-Col V positive sera (anti-ColV IgG) was adsorbed with α1(V) chain (anti-ColV IgG/ads-α1(V)) and α2(V) chain (anti-ColV IgG/ads-α2(V)) and biotinylated to evaluate the spectrum of reactivity in SSc-ILD patients lung biopsies by immunofluorescence. The SSc-ILD lung tissue samples immunostained with anti-ColV IgG showed increased green fluorescence in the vascular basement membrane, bronchiolar smooth muscle, and adventitial layer, contrasting with the tenue immunostaining in control lungs. Col V protein expression in these pulmonary compartments immunostained with early-SSc anti-ColV IgG was confirmed by immune colocalization assays with commercial anti-human Col V antibodies. In addition, SSc-ILD lung tissues immunostained with anti-ColV IgG/ads-α1(V) (sample in which Col V α1 chain-specific antibodies were removed) showed decreased green fluorescence compared to anti-ColV IgG and anti-ColV IgG/ads-α2(V). Our data show that autoimmunity to Col V in early-SSc was related to peptides of the α1(V) chain, suggesting that these antibodies could be biomarkers of SSc stages and potential target of immunotherapy with Col V immunogenic peptides.

Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, using multi-region whole exome/T cell receptor (TCR) sequencing as well as immunohistochemistry (IHC), we revealed a rather homogeneous mutational landscape but extremely cold and heterogeneous TCR repertoire in limited-stage SCLC (LS-SCLC) tumors. Compared to localized non-small cell lung cancers (NSCLCs), LS-SCLCs had similar predicted neoantigen burden and genomic ITH, but significantly colder and more heterogeneous TCR repertoire associated with higher chromosomal copy number aberration (CNA) burden. Higher mutational burden, higher T cell infiltration and positive PD-L1 expression were associated with longer overall survival (OS), while higher CNA burden were associated with shorter OS in patients with LS-SCLC.

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations., Tumor extracellular matrix has been associated with cancer progression, therapy resistance and immune suppression. Here, the authors show that collagen generates resistance to PD-1/PD-L1 immunotherapy by upregulating LAIR1 expression and downstream signaling, leading to increased CD8+ T cell exhaustion.

Small-cell lung cancer (SCLC) is speculated to harbor complex genomic intratumor heterogeneity (ITH) associated with high recurrence rate and suboptimal response to immunotherapy. Here, we revealed a rather homogeneous mutational landscape but extremely suppressed and heterogeneous T cell receptor (TCR) repertoire in SCLCs. Higher mutational burden, lower chromosomal copy number aberration (CNA) burden, less CNA ITH and less TCR ITH were associated with longer overall survival of SCLC patients. Compared to non-small cell lung cancers (NSCLCs), SCLCs had similar predicted neoantigen burden and mutational ITH, but significantly more suppressed and heterogeneous TCR repertoire that may be associated with higher CNA burden and CNA ITH in SCLC. Novel therapeutic strategies targeting CNA could potentially improve the tumor immune microenvironment and response to immunotherapy in SCLC.

Summary Background Non-small-cell lung cancer (NSCLC) is terminal in most patients with locally advanced stage disease. We aimed to assess the antitumour activity and safety of neoadjuvant chemoimmunotherapy for resectable stage IIIA NSCLC. Methods This was an open-label, multicentre, single-arm phase 2 trial done at 18 hospitals in Spain. Eligible patients were aged 18 years or older with histologically or cytologically documented treatment-naive American Joint Committee on Cancer-defined stage IIIA NSCLC that was deemed locally to be surgically resectable by a multidisciplinary clinical team, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received neoadjuvant treatment with intravenous paclitaxel (200 mg/m2) and carboplatin (area under curve 6; 6 mg/mL per min) plus nivolumab (360 mg) on day 1 of each 21-day cycle, for three cycles before surgical resection, followed by adjuvant intravenous nivolumab monotherapy for 1 year (240 mg every 2 weeks for 4 months, followed by 480 mg every 4 weeks for 8 months). The primary endpoint was progression-free survival at 24 months, assessed in the modified intention-to-treat population, which included all patients who received neoadjuvant treatment, and in the per-protocol population, which included all patients who had tumour resection and received at least one cycle of adjuvant treatment. Safety was assessed in the modified intention-to-treat population. This study is registered with ClinicalTrials.gov , NCT03081689 , and is ongoing but no longer recruiting patients. Findings Between April 26, 2017, and Aug 25, 2018, we screened 51 patients for eligibility, of whom 46 patients were enrolled and received neoadjuvant treatment. At the time of data cutoff (Jan 31, 2020), the median duration of follow-up was 24·0 months (IQR 21·4–28·1) and 35 of 41 patients who had tumour resection were progression free. At 24 months, progression-free survival was 77·1% (95% CI 59·9–87·7). 43 (93%) of 46 patients had treatment-related adverse events during neoadjuvant treatment, and 14 (30%) had treatment-related adverse events of grade 3 or worse; however, none of the adverse events were associated with surgery delays or deaths. The most common grade 3 or worse treatment-related adverse events were increased lipase (three [7%]) and febrile neutropenia (three [7%]). Interpretation Our results support the addition of neoadjuvant nivolumab to platinum-based chemotherapy in patients with resectable stage IIIA NSCLC. Neoadjuvant chemoimmunotherapy could change the perception of locally advanced lung cancer as a potentially lethal disease to one that is curable. Funding Bristol-Myers Squibb, Instituto de Salud Carlos III, European Union's Horizon 2020 research and innovation programme.

3125 Background: The Cancer Immune Monitoring and Analysis Centers Cancer Immunology Data Commons (CIMAC-CIDC) network is a NCI Cancer Moonshots initiative to provide state-of-the-art technology and expertise for immunotherapy clinical trials. Multiplex tissue immunostaining is an integral assay provided that examines density and spatial distribution of immune cells and markers in tissues, for their prognostic or predictive value. Two approaches were evaluated for sensitivity, specificity, and reproducibility and subsequently harmonized: chromogenic-based Multiplex Immunohistochemical Consecutive Staining on Single Slide (MICSSS) and Multiplex Immunofluorescence (mIF) based tyramide signal amplification system. Methods: Harmonization was performed across CIMACs (Mount Sinai, Dana Farber Cancer Institute, MD Anderson Cancer Center) in multiple steps to prove that comparable data can be generated independent of site and platform. Goals: 1) harmonize image analysis platforms alone using tissues pre-stained with single chromogenic IHC for CD3 (membrane), Ki67 (nuclear), and CD68 (cytoplasmic), 2) compare image acquisition platforms, 3) streamline Antibody (Ab) clones and assess PD-L1 detection in relation to CLIA- assays, 4) harmonize staining protocols, image acquisition, and analysis platforms on 2 test head and neck tumor samples using MICSSS and mIF, 5) validate harmonization results with a tissue microarray on 27 tissues representing multiple tumors. For last steps, each CIMAC used their platforms for PD-L1, PD-1, CD3, CD8, and pan-cytokeratin (PanCK) staining on one of three consecutive slides from serial sections and compared densities of each marker. Results: Variables as PD-1 Ab clone, positive control reference tissues, sigma value for nuclear segmentation, and use of machine-learning based cell classifier were found to be key to produce accurate, reliable, comparable data. After visual quality control assessment and comparisons of each Region Of Interest (ROI), an overall inter-site Spearman correlation coefficient of ≥0.85 was achieved per marker within each tissue and across tissue types (expect pan-Cytokeratin, ≥0.7), with average coefficient of variation ≤0.1. Conclusions: These results show for the first time that two platforms can deliver harmonized data, despite differences in protocols, platforms, reagents, and analysis tools. Data resulting from retrospective and prospective CIMAC-CIDC analyses may be used with confidence for statistical associations with clinical parameters and outcome.

Cancer cells function as primary architects of the tumor microenvironment. Yet, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblasts (CAFs) are distinguishable on the basis of gene expression signatures they acquire in co-culture with epithelial- or mesenchymal-like lung adenocarcinoma (LUAD) cells. High expression of the EMT activator ZEB1 endows LUAD cells with the capacity to activate a soluble factor exchange that leads to CAF reprogramming, to generate CAF-led invasive projections in multicellular aggregates, and to respond to pro-metastatic signals from CAFs in mice. Thus, ZEB1-expressing LUAD cells are positioned at the apex of a signaling hierarchy in the tumor microenvironment.

We report the first case series of immune checkpoint inhibitors (ICI)-associated colitis successfully treated with fecal microbiota transplantation, with reconstitution of the gut microbiome and a relative increase in the proportion of regulatory T-cells within the colonic mucosa. These preliminary data provide evidence that modulation of the gut microbiome may abrogate ICI-associated colitis.

To develop and validate a radiomics signature that can predict the clinical outcomes for patients with stage I non-small cell lung cancer (NSCLC).We retrospectively analyzed contrast-enhanced computed tomography images of patients from a training cohort (n = 147) treated with surgery and an independent validation cohort (n = 295) treated with stereotactic ablative radiation therapy. Twelve radiomics features with established strategies for filtering and preprocessing were extracted. The random survival forests (RSF) method was used to build models from subsets of the 12 candidate features based on their survival relevance and generate a mortality risk index for each observation in the training set. An optimal model was selected, and its ability to predict clinical outcomes was evaluated in the validation set using predicted mortality risk indexes.The optimal RSF model, consisting of 2 predictive features, kurtosis and the gray level co-occurrence matrix feature homogeneity2, allowed for significant risk stratification (log-rank P.0001) and remained an independent predictor of overall survival after adjusting for age, tumor volume and histologic type, and Karnofsky performance status (hazard ratio [HR] 1.27; P2e-16) in the training set. The resultant mortality risk indexes were significantly associated with overall survival in the validation set (log-rank P = .0173; HR 1.02, P = .0438). They were also significant for distant metastasis (log-rank P.05; HR 1.04, P = .0407) and were borderline significant for regional recurrence on univariate analysis (log-rank P.05; HR 1.04, P = .0617).Our radiomics model accurately predicted several clinical outcomes and allowed pretreatment risk stratification in stage I NSCLC, allowing the choice of treatment to be tailored to each patient's individual risk profile.

KRAS is the most common oncogenic driver in lung adenocarcinoma (LUAC). We previously reported that STK11/LKB1 (KL) or TP53 (KP) comutations define distinct subgroups of KRAS-mutant LUAC. Here, we examine the efficacy of PD-1 inhibitors in these subgroups. Objective response rates to PD-1 blockade differed significantly among KL (7.4%), KP (35.7%), and K-only (28.6%) subgroups (P < 0.001) in the Stand Up To Cancer (SU2C) cohort (174 patients) with KRAS-mutant LUAC and in patients treated with nivolumab in the CheckMate-057 phase III trial (0% vs. 57.1% vs. 18.2%; P = 0.047). In the SU2C cohort, KL LUAC exhibited shorter progression-free (P < 0.001) and overall (P = 0.0015) survival compared with KRASMUT;STK11/LKB1WT LUAC. Among 924 LUACs, STK11/LKB1 alterations were the only marker significantly associated with PD-L1 negativity in TMBIntermediate/High LUAC. The impact of STK11/LKB1 alterations on clinical outcomes with PD-1/PD-L1 inhibitors extended to PD-L1–positive non–small cell lung cancer. In Kras-mutant murine LUAC models, Stk11/Lkb1 loss promoted PD-1/PD-L1 inhibitor resistance, suggesting a causal role. Our results identify STK11/LKB1 alterations as a major driver of primary resistance to PD-1 blockade in KRAS-mutant LUAC. Significance: This work identifies STK11/LKB1 alterations as the most prevalent genomic driver of primary resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. Genomic profiling may enhance the predictive utility of PD-L1 expression and tumor mutation burden and facilitate establishment of personalized combination immunotherapy approaches for genomically defined LUAC subsets. Cancer Discov; 8(7); 822–35. ©2018 AACR. See related commentary by Etxeberria et al., p. 794. This article is highlighted in the In This Issue feature, p. 781

BackgroundOral squamous cell carcinomas (OSCC) prognosis remains poor. While AJCC TNM 8th edition has slightly improved patients‘ stratification with regard to prognostic, innovative approaches to are still needed. As in other tumor types, tumor immune microenvironment (TiME) might represent an opportunity to improve prognostic assessment.MethodsTiME landscape of 47 HPV-negative OSCC was analyzed using multiplex immunofluorescence (mIF). Markers for tumor cells (PanCK), tumor infiltrating lymphocytes (CD3, CD8), macrophages (CD68), inhibitory (PD-1, PD-L1, TIM3, LAG3, VISTA) or stimulatory (OX40, ICOS) immune checkpoints (ICP) were studied. Regions of interest (ROI), 5 in the tumor core and 5 at the invasion front, were subjected to cell markers identification and quantification (scoring) as well as tissue compartmentalization to divide them in tumor-epithelial and tumor-stroma compartments, respectively. A total of 20 cell phenotypes were defined based on previous work (CK+, CK+PD-L1+, CD3+, CD3+CD8+, CD3+PD-1+, CD3+CD8+PD-1+, CD3+PD-L1+, CD3+CD8+PD-L1+, CD3+PD-L1+PD-1+, CD3+CD8+PD-L1+PD-1+, CD68+, CD68+PD-L1+, CK+OX40+, CD3+VISTA+, CD3+ICOS+, CD3+LAG3+, CD3+OX-40+, CD3+TIM3+). Results were correlated with clinical features including disease-specific survival (DSS) using the Kaplan-Meier method and a multivariate Cox model. A multivariate general linear model (GLM) was built to test the specific association of each variable with a given cell density by correcting the possible confusion due to other variables.ResultsImmune cells densities were significantly higher overall in the stroma. The intra-tumor stroma showed a significant enrichment of in CD3+PD-1+ T cells compared to peri-tumor stroma. None of the clinical or pathological (resection margin, tumor stage, lymph node invasion, perineural invasion) was significantly associated with DSS. In contrast, the following cell phenotypes in the tumor invasion front were strongly associated with a poor DSS, including CD3+PD-L1+ (P-value= 0.004), CD3+PD1+PD-L1+ (P-value= 0.02) and CD3+OX40+ (P-value= 0.02) T cells as well as CD3+CD8+PD-1+ (P-value= 0.048), CD3+CD8+PD-L1+ (P-value= 0.008) and CD3+CD8+PD1+PD-L1+ (P-value= 0.01) cytotoxic T cells. In the tumor core, CD68+PD-L1- macrophages (P-value= 0.06) were marginally associated with better DSS. Using a GLM, we found that tumor from smoker-drinker patients and/or with pN+, were significantly more infiltrated by PD-1- and/or PD-L1-positive immune cells. On the other hand, floor of mouth and gingiva-mandibular OSCC were significantly less infiltrated than others.ConclusionsThe prognostic value of PD-1+ and/or PD-L1+ cells in the invasion front of resected OSCC was remarkable, underlying the importance of this area when studying the TiME. Incorporating TiME analysis in the invasion front may improve prognostic evaluation of patients treated for OSCC, especially in the context of immunotherapy.AcknowledgementsThis study was supported by a strategic alliance between the Translational Molecular Pathology-Immunoprofiling las (TMP-IL) at the Department Translational Molecular Pathology, the University of Texas MD Anderson Cancer Center and the Université Claude Bernard Lyon, Centre de Recherche en Cancérologie de Lyon and the Department of Translational Medicine, Centre Léon Bérard, Lyon, France. The authors would acknowledge ITMO Cancer 2020, ”Formation à la Recherche Fondamentale et Translationnelle en Cancérologie” (JB); CLARA 2020 ”Soutien à la mobilité des jeunes chercheurs en oncologie, N° CVPPRCAN000198” (JB); Fondation de France 2020 ”Aide à la mobilité international de médecins et pharmaciens, N° 00112162” (JB); Ligue contre le cancer 2021, comité de Saône-et-Loire (PS); 2017-INCa-DGOS-Inserm_12563: INCa SIRIC-LYriCAN INCa-DGOS-Inserm_12563 (PS)Ethics ApprovalThe study was conducted in accordance with all applicable laws, rules, and requests of French and European government authorities. Written informed consent was obtained from all patients and the study was approved by the Centre Leon Bérard institutional review board (Lyon, France). Samples were obtained from the CRB Centre Léon Bérard (n°BB-0033-00050) which is quality certified according NFS96-900 French standard and ISO 9001 for clinical trials.ConsentWritten informed consent was obtained from all patients and the study was approved by the Centre Leon Bérard institutional review board (Lyon, France)

BackgroundHuman papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HPV+ HNSCC) is a disease that has moderate response to anti-PD-1/L1 immune checkpoint blockade, with the response rates less than 20% and median progression-free survival less than 3 months. A greater understanding of tumor intrinsic and extrinsic factors that restrict anti-tumor immunity in the tumor immune microenvironment (TIME) is needed to identify other immune checkpoints to enhance therapeutic efficacy.MethodsTwo cohorts (TCGA n=72 and a separate cohort n=84) of surgically resected, treatment-naïve HPV+ HNSCC with RNA-seq were analyzed to understand the immune features. In addition, single-cell RNA-seq and TCR-seq were performed on 18 cases to further delineate the immune molecules' interactions. An immune-competent murine HPV+ HNSCC model was used to preliminarily evaluate the therapeutic efficacy.ResultsIn two bulk-sequenced HPV+ HNSCC cohorts, TIGIT ligands PVR and NECTIN2 were found to associate with an epithelial-to-mesenchymal gene expression signature, suppression of IFNα and IFNγ signaling, a stromal-enriched or immune-excluded TIME, and poor survival. Single-cell RNA-seq of over 72,000 cells of HPV+ HNSCC revealed that the PVR/NECTIN ligand TIGIT was highly prevalent in T-cells (34%), significantly higher than PD1- (20%, pConclusionsTIGIT-PVR/NECTIN receptors/ligands are more abundant than PD-1/L1 in the TIME of HPV+ HNSCC. Co-blockade of TIGIT and PD-1 immune checkpoints enhanced anti-tumor efficacy in a CD8+ T-cell-dependent manner and conferred long-term immune protection in a murine model. Our study nominates TIGIT as a therapeutic target for HPV+ HNSCC.

Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity. Significance: The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. Cancer Discov; 7(10); 1088–97. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047

Programmed cell death ligand (PD-L1) has been studied as a predictive immunotherapy biomarker. We investigated PD-L1 expression in the whole tumor and in tumor-infiltrating macrophages (TIMs) as a prognostic biomarker in surgically resected pathologic stage I non-small cell lung cancer. Pathologic specimen from 113 patients with stage I lung cancer (pT1-2a, N0, M0, tumor size 1-5 cm, 79 adenocarcinoma, 34 squamous cell carcinoma) were analyzed for PD-L1 expression in the tumor and in the TIMs using immunohistochemistry and image analysis. Statistics included recursive partitioning, univariable, multivariable, and Kaplan-Meier analyses. Patients whose tumors expressed4.7% PD-L1 (N = 87) experienced significantly better overall survival (OS) (P = 0.001) than patients with PD-L14.7% (N = 26). Patients with PD-L1 expression in macrophages 6.3% (N = 24) also experienced significantly better (P = 0.005) OS than patients with6.3% (N = 89). The best outcomes were observed in patients with low PD-L1 expression in both tumor and macrophages with 5-year OS of 94% (N = 17). Contrarily, patients with high PD-L1 expression in both tumor and macrophages experienced 5-year OS of 20% (N = 19). Low PD-L1 expression in the tumor and in the TIMs was independently associated with survival in multivariable analysis (P = 0.000 and P = 0.030, respectively). Lower PD-L1 % expression in the tumor and in the TIMs seems to be associated with significantly better OS in surgically resected stage I lung cancer. Additional studies are needed to validate PD-L1 as a prognostic biomarker in lung cancer and to study the mechanisms of intratumoral immune response.

Multiplexed imaging platforms to simultaneously detect multiple epitopes in the same tissue section emerged in the last years as very powerful tools to study tumor immune contexture. These revolutionary technologies are providing a deep methodology for tumor evaluation in formalin-fixed and paraffin-embedded (FFPE) to improve the understanding of tumor microenvironment, new targets for treatment, prognostic and predictive biomarkers, and translational studies. Multiplexed imaging platforms allow for the identification of several antigens simultaneously from a single tissue section, core needle biopsies, and tissue microarrays. In recent years, multiplexed imaging has improved the abilities to characterize the different types of cell populations in malignant and non-malignant tissues, and their spatial distribution in relationship to clinical outcomes. Multiplexed technologies associated with digital image analysis software offer a high-quality throughput assay to study cancer specimens at multiple time points before, during and after treatment. The aim of this chapter is to provide a review of multiplexed imaging covering its fundamentals, advantages, disadvantages, and material and methods for staining applied to FFPE tumor tissues and focusing on the use of multiplex immunofluorescence with tyramine signal amplification staining for immune profiling and translational research.

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8

Made available in DSpace on 2019-10-06T16:50:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-07-24. Added 1 bitstream(s) on 2019-10-09T18:35:39Z : No. of bitstreams: 1 S2523-31062019000100222.pdf: 3173113 bytes, checksum: 0b6f55fa648748e9f3293c96d1a42acb (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) BACKGROUND: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. METHODS: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p

Non-small-cell lung cancer (NSCLC) demonstrates remarkable molecular diversity. With the completion of The Cancer Genome Atlas (TCGA), there is opportunity for systematic analyses of the entire TCGA NSCLC cohort, including comparisons and contrasts between different disease subsets. On the basis of multidimensional and comprehensive molecular characterization (including DNA methylation and copy, and RNA and protein expression), 1023 NSCLC cases-519 from TCGA adenocarcinoma (AD) project and 504 from TCGA squamous cell carcinoma (SQCC) project-were classified using a 'cluster-of-clusters' analytic approach. Patterns from TCGA NSCLC subsets were examined in independent external databases, including the PROSPECT (Profiling of Resistance patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax) NSCLC data set. Nine genomic subtypes of NSCLC were identified, three within SQCC and six within AD. SQCC subtypes were associated with transcriptional targets of SOX2 or p63. One predominately AD subtype (with a large proportion of SQCC) shared molecular features with neuroendocrine tumors. Two AD subtypes manifested a CpG island methylator phenotype. Three AD subtypes showed high p38 and mTOR pathway activation. AD subtypes associated with low differentiation showed relatively worse prognosis. SQCC subtypes and two of the AD subtypes expressed cancer testis antigen genes, whereas three AD subtypes expressed several immune checkpoint genes including PDL1 and PDL2, corresponding with patterns of greater immune cell infiltration. Subtype associations for several immune-related markers-including PD1, PDL1, CD3 and CD8-were confirmed in the PROSPECT cohort using immunohistochemistry. NSCLC molecular subtypes have therapeutic implications and lend support to a personalized approach to NSCLC management based on molecular characterization.

Purpose: Promising results in the treatment of non–small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. Results: Epithelial–mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630–42. ©2016 AACR. See related commentary by Datar and Schalper, p. 3422

Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used KrasLA1 mice, which develop lung adenocarcinomas from somatic activation of a KrasG12D allele. The lung tumors in KrasLA1 mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde–derived collagen cross-links (HLCC) and lower lysine-aldehyde–derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. Implications: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells. Mol Cancer Res; 14(3); 287–95. ©2015 AACR . This article is featured in Highlights of This Issue, [p. 239][1] [1]: /lookup/volpage/14/239?iss=3

Purpose: We previously demonstrated the association between epithelial-to-mesenchymal transition (EMT) and drug response in lung cancer using an EMT signature derived in cancer cell lines. Given the contribution of tumor microenvironments to EMT, we extended our investigation of EMT to patient tumors from 11 cancer types to develop a pan-cancer EMT signature. Experimental Design: Using the pan-cancer EMT signature, we conducted an integrated, global analysis of genomic and proteomic profiles associated with EMT across 1,934 tumors including breast, lung, colon, ovarian, and bladder cancers. Differences in outcome and in vitro drug response corresponding to expression of the pan-cancer EMT signature were also investigated. Results: Compared with the lung cancer EMT signature, the patient-derived, pan-cancer EMT signature encompasses a set of core EMT genes that correlate even more strongly with known EMT markers across diverse tumor types and identifies differences in drug sensitivity and global molecular alterations at the DNA, RNA, and protein levels. Among those changes associated with EMT, pathway analysis revealed a strong correlation between EMT and immune activation. Further supervised analysis demonstrated high expression of immune checkpoints and other druggable immune targets, such as PD1, PD-L1, CTLA4, OX40L, and PD-L2, in tumors with the most mesenchymal EMT scores. Elevated PD-L1 protein expression in mesenchymal tumors was confirmed by IHC in an independent lung cancer cohort. Conclusions: This new signature provides a novel, patient-based, histology-independent tool for the investigation of EMT and offers insights into potential novel therapeutic targets for mesenchymal tumors, independent of cancer type, including immune checkpoints. Clin Cancer Res; 22(3); 609–20. ©2015 AACR.

4084 Background: Consensus Molecular Subtype 4 (CMS4) colorectal cancer (CRC) features increased TGFβ signaling, which may account for de novo resistance to immunotherapy for patients (pts) with microsatellite stable mCRC. To date, no prior trial has incorporated CMS status as an integral biomarker. Bintrafusp alfa (M7824) is a dual PD-L1 antibody/TGFβ trap with acceptable safety. Methods: Primary tumors from pts with metastatic CRC underwent CMS testing in a CLIA setting. In this Simon two-stage phase II trial (Ho: p < .05; Ha: p≥.25) for CMS4 mCRC, pts received bintrafusp alfa 1200mg IV every 14 days. RT (8Gy/day x 3 days) to a single metastatic lesion with abscopal intent was administered between doses 2 and 3. The primary objective was to estimate response rate (RR) per iRECIST. Correlative studies including RNA sequencing were performed on pre- and on-treatment biopsies. Results: 53 of 137 tested pts (39%) between June 2018-December 2019 had CMS4 mCRC. 13 of 15 treated pts received the agent with RT. All pts were evaluable for toxicity, and 13 for response. Median number of doses was 3 (IQR, 2-4). There was one grade 3 immune-related adverse event (colitis) requiring study discontinuation. There were 2 pts with stable disease and 11 with progressive disease as best response (RR 0%, 95% CI 0-22%). Enrollment was stopped after first stage for futility. Median PFS and OS were 1.6 months and 5.0 months, respectively. In paired samples, treatment with bintrafusp alfa resulted in an increase in the expression of IFNγ signature in nonirradiated metastatic lesions ( p< .001, q< .001). Updated results will be presented. Conclusions: This is the first reported clinical trial to utilize CMS status as an integral biomarker for pts with metastatic CRC and capitalizes on treating CRC subpopulations with targeted agents based upon validated RNA-based signatures. Although the efficacy for bintrafusp alfa and RT is low, changes in IFNγ signature provides a potential signal for refining therapeutic strategies based upon TGFβ enrichment in pts with mCRC. Clinical trial information: NCT03436563 .

509 Background: In TNBC patients (pts) receiving NAST, increasing tumor infiltrating lymphocytes (TILs) is associated with higher pathologic complete response (pCR) rates. However, since the presence of TIL do not consistently predict pCR, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Methods: T cell receptor (TCR) sequencing, PD-L1 immunohistochemistry and multiplex immunofluorescence were performed on prospectively collected pre-NAST tumor samples from 98 pts with stage I-III TNBC enrolled in ARTEMIS (NCT: 02276443). TCR clonality was calculated using Shannon’s entropy. PD-L1+ was defined as ≥1% immune cell staining. Response to NAST was defined using the residual cancer burden (RCB) index. Associations between TCR clonality, immune phenotype, and response were examined with the Wilcoxon rank sum test, Spearman’s rank correlation and multivariable logistic regression using stepwise elimination (threshold p > 0.2), as appropriate. Results: The pCR rate was 39% (38/98). pCR was associated with higher TCR clonality (median = 0.2 [in pts with pCR] vs 0.1 [in pts with residual disease], p = 0.05). Notably, the association between pCR and higher TCR clonality was observed in pts with ≥5% TIL (n = 61; p = 0.05) but not in pts with < 5% TIL (n = 37; p = 0.87). Among pts with ≥5% TIL, TCR clonality emerged as the only independent predictor of response in a multivariable model of tumor immune characteristics (odds ratio/0.1 increase in TCR clonality: 3.0, p = 0.021). PD-L1+ status was associated with higher TCR clonality (median = 0.2 [in PD-L1+] vs 0.1 [in PD-L1-], p = 0.004). Higher TCR clonality was associated with higher CD3+ (rho = 0.32, p = 0.0018) and CD3+CD8+ (rho = 0.33, p = 0.0013) infiltration but lower expression of PD-1 on CD3+ (rho = -0.24, p = 0.021) and CD3+CD8+ cells (rho = -0.21, p = 0.037). Conclusions: In TNBC, a more clonal T cell population is associated with an immunologically active microenvironment (higher CD3+ and CD3/8+ T cell; lower PD-1+CD3+ and PD-1+CD3/8+ T cell; PD-L1+) and favorable response to NAST, especially in pts with ≥5% TIL, suggesting a role for deep immune phenotyping in further refining the predictive value of TILs.

4 Background: Penile Squamous Cell Carcinoma (PSCC) is a rare but often fatal disease. In this study, we characterize the poorly understood immune microenvironment using multiplex immunofluorescence (mIF) and image analysis approaches in 54 patients with PSCC. Methods: Representative blocks of 54 PSCC patients were stained for six immune markers: CD3, CD8, CD68, PD-1, PD-L1, Pancytokeratin and DAPI. Two experienced pathologists using an image analysis system (InForm 2.2.4) divided them into the tumor and stroma compartment and assessed the different densities of cell phenotypes using R studio with results expressed as cells/mm2. The statistical correlations were performed using Fisher’s exact test, Pearson and Log-rank test for Kaplan Meyer plots. Results: 54 patients with confirmed diagnosis of PSCC had a median age of 62 (IQR 50-70). All samples were from the primary penile tumor with the majority of cases being HPV(–) (62%). We observed significantly higher stromal cytotoxic T cells in HPV(+) cases compared to HPV(–) ( P=0.04). Using the mean macrophage count as cutoff for positivity, high densities of total tumor macrophages CD68+ were associated with significantly improved estimated median cancer specific survival (CSS) (NA, P=0.04), median overall survival (OS) (68mos vs NA P=0.02) and lower risk of regional recurrence ( P=0.04). On the other hand, the high densities of stromal cytotoxic T cells antigen-experienced (CD3+CD8+PD-1+), was associated with significantly worse median OS (27 vs 102mos P=0.05) and median disease free survival (DFS) (18.2mos vs NA P= 0.07). Also, high densities of stromal T cells antigen-experienced (CD3+PDL-1+), were associated with significantly better CSS (NA, P=0.06) and better median OS (142.1 vs 68.8mos P=0.14). Conclusions: Using novel multiplex image analysis to assess the immune microenvironment in primary PSCC, we showed that high macrophage levels were associated with lower risk of recurrence and improved survival outcomes. Moreover, a low level of exhausted stromal cytotoxic PD-1+ T cells was associated with improved PSCC survival. Further characterization of T cell subsets in relation to tumor HPV status is ongoing.

Adaptive immune cells, including CD4For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p 0.05 considered significant.Compared with the controls, SLE-induced animals presented increased numbers of CD4Increased numbers of CD4

Background Ischemia–reperfusion injury (IRI) is one of the principal obstacles for the lung transplantation (LTx) success. Several strategies have been adopted to minimize the effects of IRI in lungs, including ex vivo conditioning of the grafts and the use of antioxidant drugs, such as methylene blue (MB). We hypothesized that MB could minimize the effects of IRI in a LTx rodent model. Methods Forty rats were divided into four groups (n = 10) according to treatment (saline solution or MB) and graft cold ischemic time (3 or 6 h). All animals underwent unilateral LTx. Recipients received 2 mL of saline or MB intraperitoneally before transplantation. After 2 h of reperfusion, arterial blood and exhaled nitric oxide samples were collected and bronchoalveolar lavage performed. Then animals were euthanized, and histopathology analysis as well as cell counts and cytokine levels measurements in bronchoalveolar lavage fluid were performed. Results There was a significant decrease in exhaled nitric oxide, neutrophils, interleukin-6, and tumor necrosis factor-α in MB-treated animals. PaO2 and uric acid levels were higher in MB group. Conclusions MB was able in attenuating IRI in this LTx model.

Immune-profiling is becoming an important tool to identify predictive markers for the response to immunotherapy. Our goal was to validate multiplex immunofluorescence (mIF) panels to apply to formalin-fixed and paraffin-embedded tissues using a set of immune marker antibodies, with the Opal™ 7 color Kit (PerkinElmer) in the same tissue section. We validated and we described two panels aiming to characterize the expression of PD-L1, PD-1, and subsets of tumor associated immune cells. Panel 1 included pancytokeratin (AE1/AE3), PD-L1, CD4, CD8, CD3, CD68, and DAPI, and Panel 2 included pancytokeratin, PD-1, CD45RO, granzyme B, CD57, FOXP3, and DAPI. After all primary antibodies were tested in positive and negative controls by immunohistochemistry and uniplex IF, panels were developed and simultaneous marker expressions were quantified using the Vectra 3.0™ multispectral microscopy and image analysis InForm™ 2.2.1 software (PerkinElmer).These two mIF panels demonstrated specific co-localization in different cells that can identify the expression of PD-L1 in malignant cells and macrophages, and different T-cell subpopulations. This mIF methodology can be an invaluable tool for tumor tissue immune-profiling to allow multiple targets in the same tissue section and we provide that is accurate and reproducible method when is performed carefully under pathologist supervision.

Background Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Methods Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. Results This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Conclusion(s) Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

Objective Systemic sclerosis (SSc)–related interstitial lung disease (ILD) is one of the leading causes of mortality. We undertook this study to analyze the gene expression of lung tissue in a prospective cohort of patients with SSc-related ILD and to compare it with that in control lungs and with 2 prospective clinical parameters in order to understand the molecular pathways implicated in progressive lung disease. Methods Lung tissue was obtained by open lung biopsy in 28 consecutive patients with SSc-related ILD and in 4 controls. High-resolution computed tomography (HRCT) and pulmonary function testing (PFT) were performed at baseline and 2–3 years after treatment based on lung histologic classification. Microarray analysis was performed, and the results were correlated with changes in the HRCT score (FibMax) and PFT values. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to confirm differential levels of messenger RNA and protein. Results Lung microarray data distinguished patients with SSc-related ILD from healthy controls. In the lungs of patients with SSc-related ILD who had nonspecific interstitial pneumonia (NSIP), expressed genes included macrophage markers, chemokines, collagen, and transforming growth factor β (TGFβ)– and interferon (IFN)–regulated genes. Expression of these genes correlated with progressive lung fibrosis defined by the change in FibMax. Immunohistochemistry confirmed increased markers of collagen (COL1A1), IFN (OAS1 and IFI44), and macrophages (CCL18 and CD163), and the positive correlation with the change in FibMax was confirmed by qPCR in a larger group of SSc patients with NSIP. Several genes correlated with both the change in FibMax (r > 0.4) and the change in % predicted forced vital capacity (r < −0.1), including IFN and macrophage markers, chemokines, and heat-shock proteins. Conclusion These results highlight major pathogenic pathways relevant to progressive pulmonary fibrosis in SSc-related ILD: macrophage emigration and activation, and up-regulated expression of TGFβ- and IFN-regulated genes.

e14047 Background: Elevated neutrophil-to-lymphocyte ratio (NLR) has been associated with poor prognosis in non-small cell lung cancer (NSCLC); the biological underpinnings of this observation have not been fully elucidated. We examined the relationships between peripheral neutrophil counts (PMN), NLR, circulating cytokines and angiogenic factors (CAF), and tumor microenvironment (TME) features in NSCLC. Methods: 150 patients with resectable NSCLC were enrolled in an immunoprofiling project. A panel of 43 CAFs was used to analyze preoperative plasma samples. Chemotherapy-naïve patients with CAF and a complete blood count ≤30 days preoperatively were included (n = 66; Table). For a subset, transcriptional signatures (MCP-counter, n = 50) and flow cytometry (n = 19) were used to identify TME phenotypes. Results: Increased PMNs were associated with increased pro-inflammatory CAF such as IL-1b (r = 0.392) and IL-6 (r = 0.339), as well as Th17/Tc17 associated CAF IL-17A (r = 0.320) and TNF-a (r = 0.368). Elevated NLR was inversely correlated with the lymphocyte activation marker soluble CD27 (r = -0.320, p = 0.009). This negative association was mirrored in the TME, as tumor neutrophil signatures were inversely correlated with a local IFN-g gene signature (r = -0.626, p < 0.001). Interestingly, a Th17/Tc17 peripheral signature (elevated IL-17A) was associated with an enrichment of CD8+TIM3+ cells (r = 0.623, p = 0.042) in the tumor. While this requires confirmation in a larger cohort, this correlation provides a potential rationale for targeting TIM3 in this population. Upon analysis of clinical characteristics, peripheral PMNs and NLR were higher among patients with squamous histology (PMN p = 0.009; NLR p = 0.034) and positively correlated with tumor size (PMN r = 0.344, p = 0.004; NLR r = 0.363, p = 0.003). Conclusions: A relative neutrophilia in NSCLC patients is associated with an inflammatory milieu suggestive of a Th17/Tc17 presence and decreased lymphocyte activation that is reflected within the TME. Further investigation is needed to define the role of NLR as a predictive biomarker and to identify whether neutrophils or Th17/Tc17 T cells could serve as a therapeutic target to improve immunotherapy response in NSCLC.[Table: see text]

585 Background: We previously reported a median tumor volume loss of 88% (range 30-98%) in 13 patients with early stage BRCA1/2 mutant breast cancer treated on a neoadjuvant trial of the PARP inhibitor talazoparib. The effects of PARP inhibition on immune aspects of the TiME in early-stage breast cancer has not been well described. The goal of this study was to evaluate the TiME in pre and post-treatment core biopsies from enrolled patients. Methods: Eleven paired core biopsies were available for examination. Tumor infiltrating lymphocytes (TILs) were quantified by H&E stained slides by a central pathologist. Specimens were assessed by multiplex immunofluorescence (mIF) using a panel of 6 biomarkers (PD-1, PD-L1, CD3, CD8, CD68 and CK) with the Opal 7-color Kit in LEICA BOND auto stainer, Vectra automated quantitative pathology imaging system and inForm software (PerkinElmer). Results: In the analyzed core biopsies, there was an increase in TILs evaluated by H&E in post-treatment compared to baseline (mean 36 vs 11%). By mIF there was an increase in CD3+ T cell and CD3+CD8+ cytotoxic T cell density in post-treatment samples compared to baseline, summarized in table. PD-L1 expression in tumor cells was rare in the cohort. There was no difference in CD3+PD-1+ or CD3+CD8+ PD-1+ lymphocytes in pre and post-treatment specimens. There was also no differences in macrophages (CD68+). Evaluation of immune phenotype and imaging response will be presented in the final analysis. Conclusions: This is the first study phenotyping the immune response to neoadjuvant talazoparib in BRCA-mutant breast cancer patients. In this small cohort, intratumoral and stromal CD3+ T cells and CD3+CD8+ cytotoxic T cells increased after two months of talazoparib. Clinical trial information: NCT02282345. [Table: see text]

8504 Background: Neoadjuvant immune checkpoint inhibitors (ICIs) induce major pathologic response (MPR) rates of 20 to 45% in resected NSCLCs. We report the results of NEOSTAR - a phase 2 trial of neoadjuvant N or NI for NSCLCs. Methods: Pts with stage I-IIIA (single N2) resectable NSCLC (AJCC 7th), PS 0-1, were randomized to N (3 mg/kg IV, D1, 15, 29) or N plus I (1 mg/kg IV, D1) followed by surgery (n = 44). Primary endpoint: MPR (≤10% viable tumor), hypothesized to be higher than MPR to induction chemotherapy historical controls. Tumor immune infiltrates and pre- & post-ICI tumor PD-L1 % were assessed by flow cytometry & IHC. Wilcoxon ranked sum test & Fisher’s exact test were used for comparisons. Results: 44 pts were randomized, 23 N, 21 NI: mean age 66, 64% males, 18% never smokers, 59% adenocarcinomas, stages: IA 8 (18%), IB 15 (34%), IIA 7 (16%) IIB 5 (11%); IIIA 9 (20%). Only 3 pts received < 3 doses due to TRAEs (7%). 34 pts had surgery post ICIs (7 not resected [7/41], 17%, [2 N, 5 NI], 3 pending). There were 10 MPRs in 41 pts overall (24%, 4 N, 6 NI), of which 6 were path CRs (15%, 2 N [9%], 4 NI [21%]). Among 34 resected pts, MPR rate was 29% (N 20%, NI 43%). Median % of viable tumor was lower post NI vs N (20% vs 65%, p = .097). ORR (RECIST v1.1) was 22% (8 PRs [5 N, 3 NI], 1 CR [NI]); 15% of pts had PD (3 N, 3 NI). The proportion of CR+PR in MPR+ was higher than in MPR- (6 [60%] vs 2 [7%], p < .001). Surgical complications included 2 bronchopleural fistulas (BPFs) in N & 8 air leaks (5 N, 3 NI). G3-G5 TRAEs included a death due to BPF post steroid-treated pneumonitis (G5, N); G3 pneumonia, hypoxia, hypermagnesemia (1 each, all N), G3 diarrhea (1 NI). CD3+ & CD103+ tissue resident memory CD8+ TILs were higher in NI- vs N-treated tumors (CD3+ 81.2% vs 54.4%, p = .028; CD8+ 56.2% vs 38.3%, p = .069). Median pre-treatment tumor PD-L1 was higher in responders (MPR+, CR+PR) vs non-responders (80% vs 1%, p = .024), and the % of viable tumor was lower in tumors with PD-L1 > 1% vs PD-L1 ≤1% (median 20% vs 80%, p = .046). Conclusions: Overall a 24% MPR rate to neoadjuvant ICIs was observed. NI induced a higher % of non-viable tumor and of tissue resident memory TILs vs N. Antitumor activity was associated with higher pre-treatment PD-L1 levels. Clinical trial information: NCT03158129.

We analyzed the effects of different administration routes and application times of the BCG-Moreau strain on airway and lung inflammation and remodeling in a murine model of allergic asthma. BALB/c mice (n=168) were divided into two groups. The first group received BCG-Moreau strain while the second group received saline using the same protocol. BCG or saline were intradermally or intranasally injected one or two months before the induction of asthma. Mice were further sensitized and challenged with ovalbumin or received saline. Twenty-four hours after the last challenge, BCG prevented the triggering of pro-inflammatory cytokines, probably by increasing Foxp3 and interleukin (IL)-10, modulating eosinophil infiltration and collagen fiber deposition, thus reducing airway hyperresponsiveness. In conclusion, BCG-Moreau prevented lung remodeling in the present model of allergic asthma, regardless of administration route and time of vaccination. These beneficial effects may be related to the increase in regulatory T cells and to IL-10 production in tandem with decreased Th2 cytokines (IL-4, IL-5, and IL-13).

Idiopathic interstitial pneumonias include complex diseases that have a strong interaction between genetic makeup and environmental factors. However, in many cases, no infectious agent can be demonstrated, and these clinical diseases rapidly progress to death. Theoretically, idiopathic interstitial pneumonias could be caused by the Epstein-Barr virus, cytomegalovirus, adenovirus, hepatitis C virus, respiratory syncytial virus, and herpesvirus, which may be present in such small amounts or such configuration that routine histopathological analysis or viral culture techniques cannot detect them. To test the hypothesis that immunohistochemistry provides more accurate results than the mere histological demonstration of viral inclusions, this method was applied to 37 open lung biopsies obtained from patients with idiopathic interstitial pneumonias. As a result, immunohistochemistry detected measles virus and cytomegalovirus in diffuse alveolar damage-related histological patterns of acute exacerbation of idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia in 38 and 10% of the cases, respectively. Alveolar epithelium infection by cytomegalovirus was observed in 25% of organizing pneumonia patterns. These findings were coincident with nuclear cytopathic effects but without demonstration of cytomegalovirus inclusions. These data indicate that diffuse alveolar damage-related cytomegalovirus or measles virus infections enhance lung injury, and a direct involvement of these viruses in diffuse alveolar damage-related histological patterns is likely. Immunohistochemistry was more sensitive than the histological demonstration of cytomegalovirus or measles virus inclusions. We concluded that all patients with diffuse alveolar damage-related histological patterns should be investigated for cytomegalovirus and measles virus using sensitive immunohistochemistry in conjunction with routine procedures.

Because histopathological changes in the lungs of patients with systemic sclerosis (SSc) are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO) and plasminogen activator inhibitor-1 (PAI-1) synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP). We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS) and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS) related significantly to staining of septal cells for interleukin (IL)-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04), septal IL-13 (P=0.03), and septal basic fibroblast growth factor (bFGF; P=0.02). Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.

Background Tacrolimus and mycophenolate have now become the most widely used combination for maintenance immunosuppressive regimens after lung transplantation in comparison with cyclosporine and azathioprine. However, limited information is available with respect to their effects on cells, other than those from the immunologic compartment. We hypothesized that different triple therapies could have different effects on airway mucociliary clearance, playing an important role in respiratory infections observed after lung transplantation. Methods Ninety rats were assigned to three groups (n = 30 each): control = vehicle, therapy 1 = tacrolimus + mycophenolate + prednisone, and therapy 2 = cyclosporine + azathioprine + prednisone. After 7, 15, or 30 days of treatment by gavage, the animals were killed and the following parameters were studied: mucus transportability, ciliary beating frequency, mucociliary transport velocity, and neutral and acid mucus production. Results There was a significant decrease in ciliary beating frequency, mucociliary transport velocity, and neutral mucus production in all immunosuppressed animals; indeed, both therapies, mainly therapy 1, caused an increase in acid mucus production for as long as 15 days of treatment. Conclusions Both triple therapies impaired airway mucociliary clearance of rats, but therapy 1 had a more deleterious effect. These data suggest that these undesirable effects can contribute to the high incidence of respiratory infections observed in patients undergoing lung transplantation.

The pathophysiologic characteristics of primary hyperhidrosis are not well understood and seem to be related to a sympathetic nervous system dysfunction. The resection of thoracic sympathetic chain ganglia is the most effective treatment for hyperhidrosis; however sympathetic ganglia function in normal individuals and in patients with hyperhidrosis is unknown.A cross-sectional study, in which 2 groups of 20 subjects were analyzed: the hyperhidrosis group (HYP), comprised of patients with hyperhidrosis who were eligible for thoracic sympathectomy, and the control group (CON) comprised of brain-dead organ donors without a history of hyperhidrosis. For each subject, the following were performed: resection of the third left sympathetic ganglion, measurement of the ganglion's diameter, and immunohistochemical evaluation by quantification of strong and weak expression areas of primary antibodies against acetylcholine and alpha-7 neuronal nicotinic receptor subunit.The presence of a strong alpha-7 subunit expression area was 4.85% in patients with primary hyperhidrosis and 2.34% in controls (p0.001), whereas the presence of a weak expression area was 11.48% in the HYP group and 4.59% in the CON group (p0.001). Strong acetylcholine expression was found in 4.95% of the total area in the HYP group and in 1.19% in the CON group (p0.001), whereas weak expression was found in 18.55% and 6.77% of the HYP and CON groups, respectively (p0.001). Furthermore, diameter of the ganglia was 0.71 cm in the HYP group and 0.53 cm in the CON group (p0.001).There is a higher expression of acetylcholine and alpha-7 neuronal nicotinic receptor subunit in the sympathetic ganglia of patients with hyperhidrosis. Furthermore, the diameter of the thoracic sympathetic chain ganglia is larger in such patients.