Immune-related Markers Research Articles

Immune Profiling Panel: A Proof-of-Concept Study of a New Multiplex Molecular Tool to Assess the Immune Status of Critically Ill Patients.

BackgroundCritical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting impaired immune response. In the field of diagnosis, a gap still remains in identifying the immune profile of critically ill patients in the intensive care unit (ICU).MethodsA new multiplex immune profiling panel (IPP) prototype was assessed for its ability to semiquantify messenger RNA immune-related markers directly from blood, using the FilmArray System, in less than an hour. Samples from 30 healthy volunteers were used for the technical assessment of the IPP tool. Then the tool was clinically assessed using samples from 10 healthy volunteers and 20 septic shock patients stratified using human leukocyte antigen–DR expression on monocytes (mHLA-DR).ResultsThe IPP prototype consists of 16 biomarkers that target the immune response. The majority of the assays had a linear expression with different RNA inputs and a coefficient of determination (R2) > 0.8. Results from the IPP pouch were comparable to standard quantitative polymerase chain reaction and the assays were within the limits of agreement in Bland–Altman analysis. Quantification cycle values of the target genes were normalized against reference genes and confirmed to account for the different cell count and technical variability. The clinical assessment of the IPP markers demonstrated various gene modulations that could distinctly differentiate 3 profiles: healthy volunteers, intermediate mHLA-DR septic shock patients, and low mHLA-DR septic shock patients.ConclusionsThe use of IPP showed great potential for the development of a fully automated, rapid, and easy-to-use immune profiling tool. The IPP tool may be used in the future to stratify critically ill patients in the ICU according to their immune status. Such stratification will enable personalized management of patients and guide treatments to avoid secondary infections and lower mortality.

Abstract PR01: Three-hit model of Wilms’ tumor formation reveals immunogenic transcriptional subtypes

Abstract Wilms’ tumor is the most common kidney cancer in children. Despite advances in care, children with metastatic, anaplastic, or relapsed disease still fare poorly. Recent studies have uncovered novel types of driver mutations, including in microRNA processing genes such as DROSHA. However, there are no ways to rationally guide therapy based on mutation, as the mechanisms by which they cause cancer remain poorly defined. Thus, here we investigated how specific types of driver mutations affect gene and protein expression. To understand how these mutations drive Wilms’ tumor formation, we first recategorized known mutations and copy number changes using a new classification schema. We found four mutation classes to be mutually exclusive with each other: microRNA processing, MYCN-activating, chromatin remodeling, and RNA splicing. These mutations were not mutually exclusive with common mutations in kidney development genes or loss-of-heterozygosity/imprinting (LOH/LOI) of chr11p15. We propose that this mutational pattern implies a “three-hit” model, whereby 11p15 LOH/LOI and mutations impairing kidney development predispose to but are often not sufficient for Wilms’ tumor formation. A third mutation then transforms the transcriptome via microRNA processing, MYCN, chromatin remodeling, or splicing. To study how these “third hits” affect gene expression, we next performed gene set enrichment analysis. As expected, we found that microRNA impairment leads to overexpression of microRNA target genes, and MYCN activation drives MYC target genes. Interestingly, we also found that loss of microRNA processing correlated with expression of oxidative phosphorylation genes, which may reveal a metabolic dependency in these tumors. In addition, mutations affecting splicing led to high levels of interferon-stimulated genes. Thus, the abnormal RNA species generated by altered splicing appear to trigger the innate immune response that normally responds to viral RNA. Finally, we measured how these mutations affect protein levels using reverse-phase protein arrays. Strikingly, Wilms’ tumors with either anaplastic histology or microRNA processing mutations express high levels of immune-related markers such as PD-1, PD-L1, phospho-Stat3, and phospho-NF-kB. In summary, many Wilms’ tumors develop a total of three types of mutations: 11p15 LOH/LOI, kidney development impairment, and a third hit that reshapes the transcriptome in an oncogenic fashion. These “third hits” may affect microRNA processing, MYCN activity, chromatin remodeling, or RNA splicing, and each type of mutation has distinct effects on gene expression. In particular, mutations in microRNA processing cause aberrant overexpression of microRNA target genes, leading to metabolic reprogramming and increased immunogenicity. As a result, some Wilms’ tumor mutations have widespread effects on the transcriptome that may be susceptible to immune checkpoint blockade. This abstract is also being presented as Poster B05. Citation Format: Kenneth S. Chen, Kavita Desai, James F. Amatruda. Three-hit model of Wilms’ tumor formation reveals immunogenic transcriptional subtypes [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR01.

Prenatal exposure to perfluoroalkyl substances modulates neonatal serum phospholipids, increasing risk of type 1 diabetes

In the last decade, increasing incidence of type 1 diabetes (T1D) stabilized in Finland, a phenomenon that coincides with tighter regulation of perfluoroalkyl substances (PFAS). Here, we quantified PFAS to examine their effects, during pregnancy, on lipid and immune-related markers of T1D risk in children. In a mother-infant cohort (264 dyads), high PFAS exposure during pregnancy associated with decreased cord serum phospholipids and progression to T1D-associated islet autoantibodies in the offspring. This PFAS-lipid association appears exacerbated by increased human leukocyte antigen-conferred risk of T1D in infants. Exposure to a single PFAS compound or a mixture of organic pollutants in non-obese diabetic mice resulted in a lipid profile characterized by a similar decrease in phospholipids, a marked increase of lithocholic acid, and accelerated insulitis. Our findings suggest that PFAS exposure during pregnancy contributes to risk and pathogenesis of T1D in offspring.

Development and verification of a personalized immune prognostic feature in breast cancer.

Breast cancer is among the highest prevalent malignant tumors worldwide with a low survival ratio. Immune-related genes have great potential as prognostic indicator in many types of tumors. Therefore, we have attempted to develop immune-related gene markers to enhance the prognosis of breast cancer. 17-IRGPs signature was constructed as a newly developed prognostic indicator to predict the survival of BC patients.

Peripheral Absolute Lymphocyte Count: An Economical and Clinical Available Immune-Related Prognostic Marker for Newly Diagnosed Multiple Myeloma.

BackgroundTo find economical and clinically available immune-related prognostic markers that could predict the overall survival (OS) of newly diagnosed multiple myeloma (NDMM) in the new drug era.Material/MethodsAbsolute lymphocyte count (ALC) and absolute monocyte count (AMC) were measured in routine blood samples from 102 patients with NDMM, and the lymphocyte-monocyte ratio (LMR) was derived. All the patients were receiving bortezomib-based chemotherapy as induction treatment. Log-rank testing was used for comparing the differences between groups. Univariate and multivariate tests were used to identify prognostic markers.ResultsThe median ALC and LMR values at diagnosis were 1.43×109/L and 3.7, respectively, and served as the cutoff point. As prognostic factors, ALC, LMR, and a new staging system combining ALC and the ISS staging system (L-ISS) were expected to have a significant impact on predicting OS. Furthermore, multivariate analysis showed that ALC ≥1.43×109/L (hazard ratio [HR]: 0.223; 95% confidence interval [CI]: 0.071–0.705; P=0.011), LMR ≥3.7 (HR: 0.363; 95% CI: 0.139–0.947; P=0.038), and L-ISS late stage (HR: 1.619; 95% CI: 1.065–2.743; P=0.027) were independent predictors for OS.ConclusionsALC and LMR can serve as surrogate markers for patients’ antitumor immunity at the initial diagnosis of multiple myeloma. A new immune-related staging system, L-ISS, which combines ALC and the ISS staging system, can predict clinical outcomes in patients who are receiving bortezomib-based chemotherapy.

Viral status, immune microenvironment and immunological response to checkpoint inhibitors in hepatocellular carcinoma

Background and aimsImmune checkpoint inhibitors (ICIs) targeting the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway have clinical activity in hepatocellular carcinoma (HCC), but only a subset of...

Colonic microbiota is associated with inflammation and host epigenomic alterations in inflammatory bowel disease

Studies of inflammatory bowel disease (IBD) have been inconclusive in relating microbiota with distribution of inflammation. We report microbiota, host transcriptomics, epigenomics and genetics from matched inflamed and non-inflamed colonic mucosa [50 Crohn’s disease (CD); 80 ulcerative colitis (UC); 31 controls]. Changes in community-wide and within-patient microbiota are linked with inflammation, but we find no evidence for a distinct microbial diagnostic signature, probably due to heterogeneous host-microbe interactions, and show only marginal microbiota associations with habitual diet. Epithelial DNA methylation improves disease classification and is associated with both inflammation and microbiota composition. Microbiota sub-groups are driven by dominant Enterbacteriaceae and Bacteroides species, representative strains of which are pro-inflammatory in vitro, are also associated with immune-related epigenetic markers. In conclusion, inflamed and non-inflamed colonic segments in both CD and UC differ in microbiota composition and epigenetic profiles.

Identification and validation of tumor environment phenotypes in lung adenocarcinoma by integrative genome-scale analysis.

To comprehensively elucidate the landscape of the tumor environment (TME) of lung adenocarcinoma (LUAD), which has a profound impact on prognosis and response to immunotherapy. Using a large dataset of LUAD patients from The Cancer Genome Atlas, Gene Expression Omnibus database (GEO), and our institution (n = 1411), we estimated the infiltration pattern of 24 immune cell populations in each sample and systematically correlated the TME phenotypes with genomic traits and clinicopathologic characteristics. The LUAD microenvironment was classified into two distinct TME clusters (A and B), and a random forest classifier model was constructed. TMEcluster A was characterized by sparse distribution of immune cell infiltration, relatively low levels of immunomodulators and slightly higher mutation load. By contrast, enrichment of both cytotoxic T cells and immunosuppressor cells was observed in TMEcluster B. Moreover, several immune-related cytokines or markers including IFN-γ, TNF-β, and several immune checkpoint molecules such as PD-L1 were also upregulated in TMEcluster B. Multivariable Cox analysis revealed that the TMEcluster was an independent prognostic factor (TMEcluster B vs. A, hazard ratio = 0.68, 95% confidence interval = 0.50-0.91, p = 0.010). These findings were all externally validated in the data from the GEO database and our institution. Our findings describe a comprehensive landscape of LUAD immune infiltration pattern and integrate several previously proposed biomarkers associated with distinct immunophenotypes, thus shedding light on how tumors interact with immune microenvironment. Our results may guide a more precise immune therapeutic strategy for LUAD patients.

Acute and chronic stress differently alter the expression of cytokine and neuronal markers genes in zebrafish brain

We report the effects of acute and chronic stress on the expression of selective immune-related genes and markers of neuronal function in the brain of the zebrafish (Danio rerio). Fish were distributed into three groups: the non-stressed control group; the acute stress (AS) group, submitted to a single stressing episode; and the unpredictable chronic stress (UCS) group, submitted to two daily stressing episodes of alternating times and types of stress. The stressing protocols were applied for a period of 14 days. The UCS protocol triggered the expression of the pro-inflammatory cytokine genes IL-1β and TNF-α, the anti-inflammatory cytokine IL-10 (negative feedback from the immune system), reduction in cFOS gene expression, and caused neuro-inflammation. The AS protocol had no effect on gene expression. Altered expression of cytokine genes, as observed in our study, correlates with several pathologies associated with neuro-inflammation, and the reduction of cFOS gene expression may indicate the occurrence of reduced neuronal plasticity. Our study further extends our knowledge about the interaction of the immune system and the different forms of stress.

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood.

Immune checkpoint inhibitors, including PD-1-blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1-blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1-blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1-blocking antibodies in cancer patients.

Molecular characterization of appendiceal goblet cell carcinoid.

231 Background: Goblet cell carcinoid (GCC) is a distinct subtype of appendiceal neoplasm that exhibits unique clinical and pathologic features. There are a few reports focusing on the molecular differences between GCC and other appendiceal tumors such as adenocarcinoma and neuroendocrine tumor (NET). Methods: A total of 495 appendiceal tumor samples (53 GCCs, 428 adenocarcinomas and 14 NETs) were tested with Next-Generation Sequencing (NGS) on a 592-gene panel and immunohistochemistry (IHC). Microsatellite instability (MSI) / mismatch repair (MMR) status was tested with a combination of NGS, IHC and fragment analysis. Tumor mutational burden (TMB) was evaluated by NGS, and PD-L1 were tested by IHC (SP142). Molecular characteristics of GCCs are compared with those of adenocarcinomas and NETs, using Chi-square test. Results: The top five genes with most frequent mutation rate in GCCs were TP53 (24.0%), ARID1A (15.4%), SMAD4 (9.4%), KRAS (7.5%) and CHEK2 (4.0%). Compared to adenocarcinomas, GCCs showed significantly lower mutation rates in KRAS (7.5% vs 60.4%), GNAS (3.8% vs 34.4%), APC (1.9% vs 11.7%), while significantly higher mutation rates in CDH1 (3.8% vs 0.7%), CHEK2 (4.0% vs 0.3%), CDC73 (2.0% vs 0.0%), ERCC2 (2.0% vs 0.0%) and FGFR2 (1.9% vs 0.0%). Compared to NETs, GCCs showed significantly lower mutation rate in KRAS (7.5% vs 28.6%), APC (1.9% vs 28.6%), BRCA2 (0.0% vs 7.1%) and FANCA (0.0% vs 7.1%), with all p < 0.05. In GCCs, MSI-H/dMMR, TML-high (> 17mut/Mb) and PD-L1 expression were seen in 0.0%, 0.0% and 2.0%, respectively. No significant difference was observed in these immune-related markers’ frequency, compared to adenocarcinomas and NETs. Conclusions: GCCs had considerably distinct mutational profile compared to appendiceal adenocarcinomas and NETs. Understanding these molecular characteristics may be critical for a development of effective treatment strategy in GCC.

Expression of inositol polyphosphate 4-phosphatase type II and the prognosis of oral squamous cell carcinoma.

Inositol polyphosphate 4-phosphatase type II (INPP4B) is a phosphoinositide phosphatase that plays complex roles in the pathogenesis of different tumors. We aimed to explore the expression, clinicopathological significance, and prognostic value of INPP4B in oral squamous cell carcinoma (OSCC). Tissue microarrays that included samples from 176 primary OSCCs, 42 normal mucosae, and 69 dysplastic tissues were used for immunostaining analyses of INPP4B protein. Aperio ScanScope CS scanner and aperio quantificationsoftware were used to scan the microarrays and score the staining, respectively. We also evaluated the correlation between INPP4B expression and clinical parameters, pathological grades, node-positive status, and immune-related markers. Expression of INPP4B was statistically significantly upregulated in human primary OSCC tissues compared with dysplastic and normal tissues. Additionally, we found that patients with strong expression of INPP4B had a statistically significantly poorer overall survival than patients with weak expression of INPP4B. Furthermore, our study indicated that expression of INPP4B in OSCC was positively associated with expression of p-S6Ser235/236 , p-CADSer1859 , and certain immune checkpoints (B7-H4, Galectin-9). Therefore, INPP4B may be an independent prognostic indicator for patients with OSCC, in which it might function as an oncoprotein.

Comprehensive molecular profiling of IDH1/2 mutant biliary cancers (BC).

479 Background: Isocitrate dehydrogenases (IDH) play a key role in energetic metabolism and IDH mutations (mut) promote oncogenesis via epigenetic and genetic changes. Data addressing the molecular contexture of IDH1/2 mut in BC are lacking. We aimed to characterize the molecular profile of IDH1/2 mutant (mIDH) GI cancers with a focus on BC. Methods: 27954 GI cancer samples collected between August 2000 and July 2019 were included: 2057 BC (1159 ICC, 277 extrahepatic CC, 573 gallbladder, 48 unspecified CC), 13807 colorectal, 4183 gastric/esophageal, 3060 other. Samples were analyzed using NextGen DNA sequencing, in situ hybridization, RNA sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mut. MMR/MSI status was evaluated by a combination of IHC, Fragment Analysis and NGS. Results: mIDH frequency in BC was 10.3% (211/2057), with higher prevalence of IDH1 mut (8.2%). ICC showed the highest mut prevalence: IDH1 13.5%, IDH2 4%. Mut rates in other GI cancers types were < 1%, except for HCC (1.9%, 11/582) and small bowel (1.1%, 8/736). When compared to IDH wild type (WT), mIDH BC showed lower mut rates in TP53 (13 vs 43%), KRAS (8 vs 19%), CDKN2A (1 vs 9%), and SMAD4 (0 vs 9%), whereas PBRM1 mut were higher (14 vs 5%) ( P < .001 for all comparisons). There was a trend towards higher frequency of ARID1A and BAP1 in mIDH BC. HER2 expression and amplification rates were lower in mIDH vs WT BC (0.5 vs 3%, P = .048 and 0 vs 6%, P = .002). FGFR2 fusion was detected in 7% of WT vs 2% of mIDH BC. mIDH BC showed a lower TMB (0.7 vs 3.7%, P = .048) and a trend for lower MSI rates (0.6 vs 3%, P = .06) vs WT BC. Conversely, IDH mut were associated with higher TMB and MSI ( P < .001) and higher PD-L1 expression in other GI cancers. Conclusions: This is the largest and most extensive profiling study to investigate the molecular makeup of mIDH BC and GI tumors. Our data show distinct gene alteration patterns characterizing mIDH BC, involving genes related to chromatin remodeling and DNA repair, and a differential expression of immune related markers compared to other mIDH GI tumors. These findings can contribute to the development of rational combination therapies and to improved patient selection in the future.

Peripheral innate immune and bacterial signals relate to clinical heterogeneity in Parkinson’s disease

The innate immune system is implicated in Parkinson’s disease (PD), but peripheral in-vivo clinical evidence of the components and driving mechanisms involved and their relationship with clinical heterogeneity and progression to dementia remain poorly explored.We examined changes in peripheral innate immune-related markers in PD cases (n = 41) stratified according to risk of developing early dementia. ‘Higher Risk’(HR) (n = 23) and ‘Lower Risk’ (LR) (n = 18) groups were defined according to neuropsychological predictors and MAPT H1/H2 genotype, and compared to age, gender and genotype-matched controls. Monocyte subsets and expression of key surface markers were measured using flow cytometry. Serum markers including alpha-synuclein, inflammasome-related caspase-1 and bacterial translocation-related endotoxin were measured using quantitative immuno-based assays. Specific markers were further investigated using monocyte assays and validated in plasma samples from a larger incident PD cohort (n = 95).We found that classical monocyte frequency was elevated in PD cases compared to controls, driven predominantly by the HR group, in whom Toll-Like Receptor (TLR)4+ monocytes and monocyte Triggering Receptor Expressed on Myeloid cells-2 (TREM2) expression were also increased. Monocyte Human Leukocyte Antigen (HLA)-DR expression correlated with clinical variables, with lower levels associated with worse cognitive/motor performance. Notably, monocyte changes were accompanied by elevated serum bacterial endotoxin, again predominantly in the HR group.Serum alpha-synuclein and inflammasome-related caspase-1 were decreased in PD cases compared to controls regardless of group, with decreased monocyte alpha-synuclein secretion in HR cases. Further, alpha-synuclein and caspase-1 correlated positively in serum and monocyte lysates, and in plasma from the larger cohort, though no associations were seen with baseline or 36-month longitudinal clinical data.Principal Components Analysis of all monocyte and significant serum markers indicated 3 major components. Component 1 (alpha-synuclein, caspase-1, TLR2+ monocytes) differentiated PD cases and controls in both groups, while Component 2 (endotoxin, monocyte TREM2, alpha-synuclein) did so predominantly in the HR group. Component 3 (classical monocytes, alpha-synuclein) also differentiated cases and controls overall in both groups.These findings demonstrate that systemic innate immune changes are present in PD and are greatest in those at higher risk of rapid progression to dementia. Markers associated with PD per-se (alpha-synuclein, caspase-1), differ from those related to cognitive progression and clinical heterogeneity (endotoxin, TREM2, TLR4, classical monocytes, HLA-DR), with mechanistic and therapeutic implications. Alpha-synuclein and caspase-1 are associated, suggesting inflammasome involvement common to all PD, while bacterial translocation associated changes may contribute towards progression to Parkinson’s dementia. Additionally, HLA-DR-associated variations in antigen presentation/clearance may modulate existing clinical disease.

High OX-40 expression in the tumor immune infiltrate is a favorable prognostic factor of overall survival in non-small cell lung cancer

IntroductionOX-40 co-stimulatory signaling plays a role in mounting anti-tumor immune responses and clinical trials targeting this pathway are ongoing. However, the association of with OX-40 protein expression with clinical outcomes and pathological features in non-small cell lung cancer (NSCLC) are largely unknown.MethodsSurgically-resected stage I-III NSCLC specimens (N = 100) were stained by immunohistochemistry (IHC) for the following immune markers: OX-40, PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FOXP3, granzyme B, and ICOS. Immune-related markers mRNA expression were also assessed. We evaluated the association of OX-40 levels with major clinicopathologic variables, including molecular driver mutations.ResultsOX-40 IHC expression was observed in all tested tumors, predominantly localized in the membrane of the tumor immune infiltrate, and was not associated with a specific clinicopathologic or molecular subtype. High OX-40 expression levels measured by IHC median score were associated with better overall survival (OS) (p = 0.002), independent of CD3/CD8, PD-L1, and ICOS expression. High OX-40 IHC score was associated with increased expression of immune-related genes such as CD3, IFN-gamma, ICOS, CD8, CXCL9, CXCL10, CCL5, granzyme K.ConclusionsHigh OX-40 IHC expression in the tumor immune infiltrate is associated with favorable prognosis and increased levels of immune-related genes including IFN-gamma in patients with surgically resected stage I-III NSCLC. Its prognostic utility is independent of PD-L1 and other common markers of immune activation. High OX-40 expression potentially identifies a unique subgroup of NSCLC that may benefit from co-stimulation with OX-40 agonist antibodies and potentially enhance the efficacy of existing immune checkpoint therapies.

Integrated Epigenetic Profiling Identifies EZH2 As a Therapeutic Target to Re-Establish Immune Recognition of Leukemia Relapses with Loss of HLA Class II Expression

Background It is becoming increasingly recognized that evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, alterations in the antigen processing and presentation machinery represent one of the most effective strategies enacted by tumor cells to avoid recognition from T cells. Whereas it is now well recognized that genomic loss of HLA is frequently at the basis of post-transplantation relapse, it was only recently reported that up to 40% of AML relapses display transcriptional downregulation and complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods Starting from primary AML samples pairwise collected from patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were analyzed for surface expression of selected immune-related markers (HLA class I and II, PD-L1, B7-H3), and characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), histone modifications associated with active promoters (H3K4me3) or regulatory elements (H3K27ac) (by ChIP-seq) and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches were integrated by Multi-Omics Factor Analysis (MOFA), followed by Gene Set Enrichment Analysis (GSEA). Finally, the immunological effects of epigenetic drugs and recombinant immune-modulatory cytokines on primary and PDX-derived AML samples were tested in ex-vivo short-term cultures on a layer of mesenchymal stromal cells. Results We verified that PDXs faithfully recapitulate immune-related differences between diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules (Figure 1A). Integration of all the high-throughput technologies by MOFA evidenced that the differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility and histone marks, and largely unrelated to the DNA methylation profile (Figure 1B). We documented that the gene sets that emerged upon integrating epigenetic analyses by MOFA matched our previously published immune-related relapse signature (Toffalori et al, Nat Med, 2019) but also and most intriguingly lists of genes known to be targeted by EZH2, the enzymatic subunit of the PRC2 chromatin repressor complex (Figure 1C). These computational analyses were supported by the evidence of a relapse-specific closed chromatin status of HLA class II genes and their regulators (Figure 1D). To revert these epigenetic changes, we inhibited EZH2 with tazemetostat (EPZ-6438), an epigenetic drug currently being tested in early-phase clinical trials for lymphomas, in two AML relapses with non-genomic loss of HLA class II expression. EZH2 inhibition reduced the levels of the repressor mark H3K27me3 (Figure 1E), increased the surface expression of HLA class II molecules on leukemia cells (Figure 1F) and ultimately improved leukemia recognition by CD4+ T cells (Figure 1G). Notably, these effects were even more pronounced when EZH2 inhibition was combined with IFN-g treatment (Figure 1F,G), suggesting synergism between this epigenetic compound and cytokines released by immune cells upon target recognition. Conclusions Our results provide mechanistic insights into epigenetic regulation of HLA class II downregulation in leukemia and a strong therapeutic rationale to test EZH2 inhibition as an innovative strategy for the treatment of AML post-transplantation relapses. Figure 1 Disclosures Vago: GenDx: Research Funding; Moderna Therapeutics: Research Funding.

Bioinformatic profiling of prognosis-related genes in the breast cancer immune microenvironment.

In the microenvironment of breast cancer, immune cell infiltration is associated with an improved prognosis. To identify immune-related prognostic markers and therapeutic targets, we determined the lymphocyte-specific kinase (LCK) metagene scores of samples from breast cancer patients in The Cancer Genome Atlas. The LCK metagene score correlated highly with other immune-related scores, as well as with the clinical stage, prognosis and tumor suppressor gene mutation status (BRCA2, TP53, PTEN) of patients in the four breast cancer subtypes. A weighted gene co-expression network analysis was performed to detect representative genes from LCK metagene-related gene modules. In two of these modules, the levels of the co-expressed genes correlated highly with LCK metagene levels, so we conducted an enrichment analysis to discover their functions. We also identified differentially expressed genes in samples with high and low LCK metagene scores. By examining the overlapping results from these analyses, we obtained 115 genes, and found that 22 of them were independent predictors of overall survival in breast cancer patients. These genes were validated for their prognostic and diagnostic value with external data sets and paired tumor and non-tumor tissues. The genes identified herein could serve as diagnostic/prognostic markers and immune-related therapeutic targets in breast cancer.

Tumor Characterization in Breast Cancer Identifies Immune-Relevant Gene Signatures Associated With Prognosis.

There has been increasing attention on immune-oncology for its impressive clinical benefits in many different malignancies. However, due to molecular and genetic heterogeneity of tumors, the activities of traditional clinical and pathological criteria are far from satisfactory. Immune-based strategies have re-ignited hopes for the treatment and prevention of breast cancer. Prognostic or predictive biomarkers, associated with tumor immune microenvironment, may have great prospects in guiding patient management, identifying new immune-related molecular markers, establishing personalized risk assessment of breast cancer. Therefore, in this study, weighted gene co-expression network analysis (WGCNA), single-sample gene set enrichment analysis (ssGSEA), multivariate COX analysis, least absolute shrinkage, and selection operator (LASSO), and support vector machine-recursive feature elimination (SVM-RFE) algorithm, along with a series of analyses were performed, and four immune-related genes (APOD, CXCL14, IL33, and LIFR) were identified as biomarkers correlated with breast cancer prognosis. The findings may provide different insights into prognostic monitoring of immune-related targets for breast cancer or can be served as reference for the further research and validation of biomarkers.

Associations of perfluoroalkyl substances with proteomic biomarkers of inflammation

PDS 62: Chemicals and metals: exposure and biomarkers, Johan Friso Foyer, Floor 1, August 28, 2019, 10:30 AM - 12:00 PM Background: Perfluoroalkyl substances (PFAS) have been linked to immunotoxicity in experimental studies. Although PFAS exposure is associated with altered immune function in epidemiological studies of children, it is less known whether these findings also hold for adults or elderly individuals. Methods: We measured a panel of 92 proteins in plasma from 965 elderly individuals from Sweden (all aged 70, 50% women) using a multiplex proximity extension assay including, among others, immune-related markers such as tumor necrosis factors, interleukins as well as cardiometabolic markers such as monocyte chemoattractant proteins. Plasma PFAS concentrations were determined using isotope-dilution ultra-pressure liquid chromatography coupled to tandem mass spectrometry. We examined associations using adjusted multivariable linear regression. Results: Of the 86 protein markers passing quality control and detection frequency >75%, 20 were negatively associated with levels of five PFAS following adjustment for sex, sample storage time in freezer, and correction for multiple testing. Associations of some PFAS and hepatocyte growth factor (HGF), macrophage colony-stimulating factor 1 (CSF-1), and tumor necrosis factor receptor 1 (TNF-R1) remained significant following further adjustment for smoking, exercise habits, education, energy, and alcohol intake. In this case, plasma concentrations of perfluorohexane sulfonic acid (PFHxS), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA) were inversely associated with HGF (βPFHxS: -0.11: 95% confidence interval (CI); -0.17, -0.06; βPFDA: -0.07: 95% CI; -0.10, -0.04, and βPFUnDA: -0.07: 95% CI; -0.11, -0.04). PFUnDA was further inversely associated with CSF-1 (β: -0.09: 95% CI; -0.12, -0.06) and TNF-R1 (β: -0.07: 95% CI; -0.10, -0.04). Conclusions: Our findings implicate PFAS exposure with decreased plasma inflammatory markers and require replication in larger studies.

1882PD - Comprehensive molecular characterization of brain metastases (BM) from colorectal cancer (CRC)

BackgroundAlthough rare, the incidence of BM is increasing due to the improvement in metastatic CRC treatment and longer survival. Genomic analyses of small series revealed that BM can harbor potentially unique driver mutations. We aimed to comprehensively characterize the molecular profile of BM and explore the differences between BM vs other distant metastases (OM) and primary tumors (PT) in CRC. MethodsTumor samples from BM (n=81), PT (n=6898) and OM (n=5918) were analyzed using NGS (TruSEQ on 45 genes or NextSEQ on 592 genes), in situ hybridization and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. ResultsThe most frequently mutated genes in BM were TP53 (80%), APC (73%), KRAS (68%), ARID1A (18%), PIK3CA (13%) and FBXW7 (9%). The most prevalent copy number increase was seen in CDX2 (20%), CCND2 (7%), FLT1 (7%), FLT3 (7%) and FOXO1 (7%). When compared to OM and PT, mutations in KRAS (BM: 68%, OM: 49%, PT: 48%), CDKN2A (5%, 1%, 1%), ERCC2 (2%, 0, 0) and HRAS (1%, 0, 0) were significantly higher in BM (P<.05); BRCA1 was significantly higher in BM vs OM (3 vs 1%, P=.025). Conversely, BRAF mutations trended to be lower in BM vs PT (4 vs 10%, P=.059). Overall, copy number alterations (CNA) were significantly higher in BM vs PT and OM, including CDX2, CCND2, FLT1, FOXO1, ERC1, FGF23, KDM5A, NSD3, FGFR1 (P<.05). HER2 overexpression showed a non-statistically significant trend to be higher in BM compared to PT and OM (7%, 2%, 3%, P=.064). Significantly lower rates of TMB-high (>17mut/MB) and MSI-high were seen in BM vs PT (2 vs 9% and 1 vs 8%, P=.049 and .031, respectively) but not compared to OM. No RSPO3 nor NTRK1 fusions were seen in BM (n=5). Female gender was associated with younger age in BM (53.5 vs 62yr, P=.0014) and OM (58.8 vs 60.2yr, P<.0001), not seen in PT. ConclusionsThis is the largest and most extensive profiling study to investigate the molecular makeup of BM and the differences with PT and OM in CRC. Our data show distinct mutations and CNA characterizing BM and lower expression of immune related markers, supporting the rationale to develop tailored approaches to the treatment of this metastatic site. Legal entity responsible for the studyThe authors. FundingNational Cancer Institute grant number P30CA014089, Gloria Borges WunderGlo Foundation-The Wunder Project, Dhont Family Foundation, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Call to Cure Research Fund and Fong Research Project. DisclosureJ. Xiu: Full / Part-time employment: Caris Life Sciences. Y. Baca: Full / Part-time employment: Caris Life Sciences. R.M. Goldberg: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. A. Grothey: Travel / Accommodation / Expenses: Caris Life Sciences. A.F. Shields: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. A. Seeber: Advisory / Consultancy: Caris Life Sciences. M.E. Salem: Travel / Accommodation / Expenses: Caris Life Sciences. P.A. Philip: Travel / Accommodation / Expenses: Caris Life Sciences. J.L. Marshall: Advisory / Consultancy: Caris Life Sciences. W..M. Korn: Full / Part-time employment: Caris Life Sciences. H.J. Lenz: Travel / Accommodation / Expenses: Caris Life Sciences. All other authors have declared no conflicts of interest.