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Introduction: Systemic immunotherapies (IO) have recently shown activity in melanoma and non-small cell lung cancer metastatic to the brain, but minimal activity in the treatment of other brain metastases. Further, only a limited percentage of melanoma or lung cancer (MBM/NSCLC)-brain metastasis (BrMs) patients respond to IO. The aim of this study is to explore the differences in the tumor microenvironment (TME) among BrMs from different tumor types including melanoma, NSCLC, breast, and renal cell carcinoma (RCC). Methods: Formalin-fixed paraffin-embedded (FFPE) tumor from 13 MBM and 43 non-MBM [18 NSCLC-BrMs, 20 breast-BrMs and 5 Renal RCC-BrMs] and primary tissue from 7 melanoma, 4 NSCLC and 3 breast cases were identified. RNA was isolated from tumor regions and subjected to whole gene expression profiling (GEP) by RNA-seq. Microenvironment Cell-Populations counter method estimated the abundance of immune and stromal infiltrated cell subpopulations. Inter- and intra-tumor heterogeneity was evaluated in selected regions of interest using a 59-plex immune related protein panel assessed by GeoMx Digital Spatial Profiling (DSP) technology. Results: Whole GEP revealed 3650 transcripts differentially expressed between MBM and non-MBM (False Discovery Rate, FDR, ≤0.01). MBM showed increased expression of genes involved in B cell function, cytolytic activity associated with CD8 and NK cells and complement signaling (C1QA/B/C). Conversely, overexpression of genes involved in epithelial signaling, cell adhesion and neurovascular coupling was observed in non MBM vs MBM. Spatial protein profiling revealed in the TME of MBM vs non-MBM, an increased expression (p 1.5) of the tumor antigen MART-1, together with a higher infiltration of CD8+ cytotoxic cells (CD8+, 41BB+), antigen presenting cells (HLA-DR+, B2M+, CD40+) and cells involved in the formation of tertiary lymphoid structures (CD20+, CD11c+). Conversely, increased infiltration of Tregs (CD25+, CD127+), neutrophils (CD66b+) and epithelial cells (EpCAM+, PanCK+) was observed in non-MBM vs MBM. Interestingly, LAG3+and PD-L2+cells were also observed to be more enriched in non-MBM vs MBM. GEP in primary tumors (FDR≤0.01) showed limited immune-related signals but revealed overexpression of genes associated with NK cells functions and immune chemoattraction (CCL18/20, CXCL2/5/17) to be overexpressed in PM vs other primary tumors. Conclusion: TME interrogation at molecular and protein levels has shown that MBM are more immune infiltrated than brain metastases derived from non-melanoma solid tumors. Particularly, a cytotoxic and B cells - enriched TME was observed in MBM vs brain metastases derived from NSCLC, breast and RCC. These findings will be validated in larger cohorts and incorporated in therapeutic investigations. Citation Format: Alberto Mendoza-Valderrey, Daria M. Kessler, Ethan Dettmann, Xinmin Li, Sierra Thompson, Steven E. Kolker, Kim A. Margolin, Maria L. Ascierto. Digital Spatial Profiler highlights a T and B cells inflamed tumor microenvironment in brain metastases derived from melanoma vs. non-melanoma solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6777.

Background: NeoAdjuvant cisplatin-based Chemotherapy (NAC) followed by cystectomy is the standard of care for patients with muscle-invasive bladder cancer (MIBC). Pathologic complete response (pCR) at the time of cystectomy predicts better survival. Unfortunately, only approximately a third of patients achieve such a response. The aim of this study is to explore in the tumor microenvironment and in the peripheral blood, biomarkers associated with pCR following NAC and to further evaluate the pharmacodynamic modifications induced by NAC on immune cells detected in the peripheral blood. Methods: N= 13 patients with MIBC received NAC and were classified as pCR (n=8) or no-pCR (n=5). Bulk RNA-seq was performed on the 13-patients formalin-fixed paraffin-embedded (FFPE) tumor biopsies collected prior to NAC treatment (baseline). Lymphocytes and neutrophils counts were assessed in the peripheral blood at baseline and following treatment with NAC. Kaplan-Meier analyses and Cox PH regression models were used for survival analyses (OS). Inter- and intra-tumor immune heterogeneity of n=59 selected immune protein was determined using GeoMx DigitalSpatial Profiling (DSP) technology. Results: Bulk RNAseq revealed 1119 transcripts differentially expressed between pCR and no-pCR (p ≤ 0.01; Abs linear Fold Change >2). Particularly, we observed an increased expression of genes involved in B cell function (CD19, CLEC4D, CXCR5), dendritic cell activation (XCR1, CCL5) and formation of tertiary lymphoid structure in the TME of pCR vs. no-pCR. On the contrary, an increased expression of IL8, DNA repair genes (POLB, DMC1, RPA4, HFM1, RAD54B) and genes involved in cell adhesion was observed at baseline in the TME of no-pCR. Interestingly, patients with higher expression of CXCR5 and XCR1 and lower expression of POLB at baseline (medium expression as cut off) were also characterized by longer overall survival following cystectomy. DSP confirmed a TLS enrichment (CD20+, HLA-DR+) in the TME of pCR prior treatment initiation. On the contrary, an increased infiltration of Tregs (CD25+, CD127+) and immunosuppressive cells (CD45+, ARG+) was detected at baseline in no-pCR. Interestingly, also an increased expression of immune checkpoints such as PD-L1, VISTA, ICOS and LAG3, was observed at baseline in the TME of no-pCR. While no clear association with NAC response was seen for lymphocytes and neutrophils counts when evaluated in baseline peripheral blood, a significant increase of lymphocytes and decrease of neutrophils counts, respectively, was observed in pCR vs no-pCR following NAC. Conclusions: When confirmed on a larger cohort, our results will lead to novel therapeutic strategies and to novel biomarkers predicting patients likely to respond to NAC. Citation Format: Maria L. Ascierto, Alberto Mendoza-Valderrey, Jane Choe, Daria M. Kessler, Xinmin Li, Jennifer A. Linehan, Przemyslaw W. Twardowski. Immune-related biomarkers associated with pathologic complete response (pCR) to neoadjuvant cisplatin-based chemotherapy in patients with muscle-invasive bladder cancer undergoing cystectomy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2167.

In recent years, immunotherapy has become a powerful therapeutic option against multiple malignancies. The unique capacity of natural killer (NK) cells to attack cancer cells without antigen specificity makes them an optimal immunotherapeutic tool for targeting tumors. Several approaches are currently being pursued to maximize the anti-tumor properties of NK cells in the clinic, including the development of NK cell expansion protocols for adoptive transfer, the establishment of a favorable microenvironment for NK cell activity, the redirection of NK cell activity against tumor cells, and the blockage of inhibitory mechanisms that constrain NK cell function. We here summarize the recent strategies in NK cell-based immunotherapies and discuss the requirement to further optimize these approaches for enhancement of the clinical outcome of NK cell-based immunotherapy targeting tumors.

ERSCongress 2019: highlights from Best Abstract awardees http://bit.ly/2XWlD7Y.

Background: Long-term survival with good allograft function (LTS) after lung transplantation (LT) is mainly limited by the development of chronic lung allograph dysfunction (CLAD). The objective of this study was to identify genes and miRNAs which might contribute to a better understanding of the molecular mechanisms influencing LTS after LT. Methods: The mRNA and miRNAs expression profiles were determined in blood samples from LTS (n=30) and CLAD patients (n=30) by microarray technology. Gene Ontology was used for enrichment analysis and the interactions between miRNAs and genes were studied by network analysis with the mixOmics R package. Results: The analysis of mRNA expression revealed that 458 genes were differentially expressed between LTS versus CLAD patients. Genes related to neutrophil-granulocyte activation and neutrophil degranulation were downregulated in LTS, suggesting a dysregulation of the innate immune system in CLAD patients. Regarding miRNAs expression analysis, 36 mature miRNAs were differentially expressed between both groups. Of these 36 elements, 30 miRNAs had experimentally validated target genes enriched in the set of 458 differentially expressed genes. Biological significance analysis showed that miRNA target genes differentially expressed between LTS and CLAD patients were related to neutrophil degranulation. Conclusion: Differences in both mRNA expression and upstream miRNA regulators have been found between LTS and CLAD patients. These results suggest that innate immune system may play a role in long-term survival after LT. Study financed by ISCIII (PI13/01076), FEDER, FUCAP, SEPAR (138/2016), Astellas, Novartis and Chiesi.

Purpose Chronic lung allograft dysfunction (CLAD) is the main limiting factor for long-term survival after lung transplantation (LT). However, there is a small number of LT patients which are long-term survivors with good allograft function (LTS). The aim of the present study was to compare the gene expression profile of CLAD and LTS patients and performing an enrichment analysis by using Reactome annotation database. Methods We analyzed whole-blood gene expression profiles of 60 double lung transplant recipients who were included in this multicenter cross-sectional study: 30 patients with CLAD and 30 patients with a stable allograft function 10 years after lung transplantation (LTS). Expression analysis were performed by microarray technology (ClariomTM D Arrays). Results We identified 458 differentially expressed genes (DEGs) between LTS and CLAD patients. Among them, 201 genes were up-regulated and 257 genes were down-regulated. Differentially expressed genes included MMP-8, LTF, TCN1 and OLFM4. Enrichment analysis showed involvement of neutrophil degranulation, class A/1 (Rhodopsin-like receptors), cell surface interaction at the vascular wall, antimicrobial peptides, interleukin-4 and 13 signaling, role of phospholipids in phagocytosis and activation of matrix metalloproteinases pathways. Conclusion It seems that neutrophil degranulation, matrix metalloproteinases and antimicrobial peptides gene expression pathways within others could be involve in differences between CLAD and LTS patients. Study financed by Instituto de Salud Carlos III (PI13/01076); the European Regional Development Fund (FEDER), FUCAP, Astellas, Novartis and Chiesi.

Chronic allograft dysfunction (CLAD) is the main limitation for Lung Transplantation (LT) survival. There is known 2 forms of CLAD: obliterative bronchiolitis syndrome (BOS) and restrictive allograft syndrome (RAS). The detection of the glycoprotein KL-6 in bronchoalveolar lavage (BAL) or serum in LT population may discriminate between both. The objective of the study was to analyse KL-6 levels in BAL and in serum from LT population in differents situations : stable (ST) , infection (LTI), BOS and RAS. Patients and methods - Forty four patients with bilateral LT and who survived more than 3 months were included. The population were divided in 4 groups : 14 ST patients, 10 LTI patients, 12 BOS patients and 8 patients with RAS. BAL and serum samples from the 44 patients were analysed with the Kit KL-6 ( Eidia Co.,Ltd.,Tokyo, Japan). U Mann Whitney were used to analysed differences between groups Results - KL-6 levels in serum were higher in RAS patients at a median of 1042 [IQR 504,9 to 1592]. Significant differences were shown between RAS vs ST, LTI and BOS patients with p-value of 0,0001, 0,0031 and 0,0055 respectively. KL-6 levels in BAL were higher in ST patients at a median of 262,3 [ IQR 117,9 to 661,8 ]. Significant differences were only shown between ST vs LTI patients (p=0,0088) and LTI vs BOS patients ( p=0,0168). Conclusion - KL-6 measured in serum from LT population with RAS are the highest values compared with the rest. KL-6 in serum seems to be better biomarker for RAS than in BAL.