e20618 Background: Immune checkpoint inhibitors (ICIs) dramatically improved clinical outcomes of patients with resectable non-small cell lung cancer (NSCLC). However, ICIs are not the scenario of “One-size fits to all”. It is of great importance to explore biomarkers, especially via liquid biopsy-based approach, to predict or monitor treatment response. Besides comprehensive genomic profiling of primary tumor tissue, this study focused on gene expression profile of PBMCs at baseline as well as one week post 1 st immunotherapy administration, and its association with major pathological response (MPR) of neoadjuvant chem-immunotherapy in resectable NSCLC patients. Methods: Pre-treatment tumor biopsies and PBMC at baseline (D0) and 7 days after neoadjuvant chem-immunotherapy (D7) were collected from 27 patients with stage IB-IIIB NSCLC from two prospective clinical trials (NCT04422392 and NCT04762030) between Aug 2019 and July 2021. Tumor biopsies were subjected for comprehensive genomic profiling by an NGS panel covering 571 cancer related genes, and RNA from PBMC was subjected to an RNA sequencing panel covering transcripts of 2660 genes. (Amoy Diagnostics, Xiamen, China). Single sample gene set enrichment analysis (ssGSEA) was used to assess enrichment of inflammatory-related gene sets. CIBERSORT and in-housed established DAISM-DNN algorithm were applied to estimate immune cell populations from PBMC-derived gene expression profile. Results: Patients received chemo-immunotherapy achieved 59.3% of MPR. Stronger signatures including effector T cell and IFN-γ/Effector T-cell were significantly enriched in baseline PBMC samples in MPR patients (p = 0.028, p = 0.042 respectively). In parallel, significant increasing Naïve CD8 positive T cells was observed in PBMC at D7 in MPR patients by both CIBERSORT (p = 0.047) and DAISM-DNN algorithm (p = 0.018). Interestingly, at D7, signature of M0 macrophage was enriched in PBMC from patients with non-MPR. In addition, comprehensive genomic profiling indicated a numerically higher level of tumor mutation burden (TMB) in pretreatment tumor samples in MPR patients (Median TMB, MPR 11.51 mut/Mb vs Non-MPR 7.19 mut/Mb, p = 0.074). Conclusions: Expression profile of baseline PBMC as well as dynamics of PBMC profile in a short period (7 days) may predict the clinical efficacy of neoadjuvant chemo-immunotherapy. More patients are being enrolled into this study. Moreover, to validate these findings, large-scale and prospective investigations are warranted. Clinical trial information: NCT04422392 and NCT04762030.
e15532 Background: Microsatellite instability (MSI) is a hypermutated phenotype primarily caused by the deficiency of DNA mismatch repair activity. Conventional MSI detection platforms are time consuming and need extra normal tissue. Due to the short DNA sequences of microsatellite loci in MSI samples, the method can be simplified to a one-step approach to differentiate MSI status by observing the melting curve. Herein a novel feasible MSI status detection method based on one step-PCR in single tumor tissue was developed, and validated in colorectal cancer samples. Methods: Specific microsatellite sites shorter than 12 bp in length (feasibility of fluorescence probes) were selected from public databases for analysis. In tumor tissues from 98 CRC patients with known MSI status, microsatellite sites with the highest receiver operating characteristic (ROC)-area under the ROC curve (AUC) were eventually selected for the final PCR panel design. The cut-off value of this assay was defined by the number of melting peaks in a specific Tm value range. An independent set of 199 tumor tissues from CRC patients with known MSI by Sanger sequencing was recruited to validate the performance of this novel PCR MSI kit. Results: The ROC-AUCs of the pre-selected eight mononucleotide repeats were 0.82, 0.93, 0.79, 0.78, 0.98, 0.95, 0.92, and 0.79. When 0 or 1 marker had a melting peak within the specified Tm value range, the sample was identified as "MSS/MSI-low (MSI-L)"; when 2 or more markers had melting peaks within the specified Tm value range, the sample was determined to be "MSI-high (MSI-H)". In the clinical sample set, compared with the results given by conventional MSI detection method, the sensitivity of the 8-gene panel for MSI-H was 99.4% (166/167), the specificity of the 8-gene panel was 100%, and the overall concordance reached to 99.5%. Conclusions: The novel PCR-MSI detection method using 8-gene panel developed in this study was feasible and accessible in clinical practice, and a large-scale clinical validation is in progress. [Table: see text]
e21051 Background: Differentiating multiple pulmonary lesions as multiple primary lung cancer (MLC) or intra-pulmonary metastasis (IPM) is critical for clinics. Radiological or histological (dis)concordance may be informative, while, genetic information may aid tracing lineage information on multiple lung lesions. This study aims to apply comprehensive genomic profiling deciphering intrinsic genetics of multiple lung lesions. Methods: 32 patients, 69 FFPE samples were applied to perform high-through put sequencing. 636 cancer-related genes were captured and sequenced by MGI-seq 2000. Raw data was processed via in-house developed pipeline. Unsupervised clustering was applied to cluster samples based on genetic characters after variant calling and annotation. Patients were categorized into “Genomic related” and “Genomic unrelated” groups. Another cohort consisting of 402 patients with mono-lesion was used as control cohort. Results: No difference of age of onset was found between 32 patients and 403 patients with mono lesion (60.5 versus 60 years). Male had relatively lower incidence of multiple lung lesions (RR:0.46,95%CI: 0.20-0.93, P = 0.04). Eight patients were classified as “Genomic related” by presenting same mutation in driver or tumor suppressor genes. Six of eight patients were characterized to carry EGFR L858R, in addition, two patients presented co-mutation of RB1(c.1814+1G > T, p.E48Kfs*18) and TP53 mutations (p.H193R, p.R249S). Another two patients carried EGFR 19 Deletion. Two lesions from one patient with predefined diagnosis of intra-pulmonary metastasis showed biallelic loss of function mutations in same loci of NF1 gene (p.S2311*/ p.Q1360*) together with same KRAS mutation (p.G13C). Radiologically, no same lobe as well as left-right preference were revealed between “Genomic related” and “Genomic unrelated” groups. Histologically, there was no enrichment of patients with same histology subtype in “genomic related” group, verse visa. While, patients in “genomic related” group had higher ages at initial diagnosis (median age 69.5 vs 56.5 years, P = 0.04). Conclusions: Comprehensive genomic profiling should be applied to clinics distinguishing the nature of multiple lung lesion irrespective of radiological and histological diagnosis. [Table: see text]
e17582 Background: PARP inhibitors (PARPi) have been widely used in the treatment of homologous recombination repair-deficient tumors. Comprehensive investigations on the resistance mechanisms of PARPi and tumor microenvironment alterations after PARPi therapy are largely insufficient. Methods: 5 ovarian cancer patients (pts) treated with PARPi at West China Second University Hospital were enrolled in this study (Ethical Lot Number: 20200076). Clinicopathological information of the pts are shown in table below. Paired PARPi treatment-naive surgical samples (TNS) and the biopsy samples that progressed after PARPi maintenance therapy (PPS) were obtained. Multi-omic profiling was conducted by an NGS panel (Master Panel, AmoyDx) containing DNA exon regions of 563 genes and RNA expression of 1831 genes analyzing genetic mutations and dynamics of gene expression profile in these paired tumor samples. Results: Median duration of treatment with PARPi was 18 months (11-22). Comprehensive genomic profiling discovered MYC amplification (n = 3), ZFHX4 mutation (n = 2) and IDH1 mutation (n = 1) in PPS. One PPS harbored simultaneous amplification of PDCD1, FGF3, MYC, FGF19 and ZFHX4 mutation. In addition, BRCA1 intron11: c.4096+1G > T mutation was detected in one PPS carrying germline BRCA1 mutation (E1066*), which putatively resulted in restoration of BRCA1 function. At RNA level, single-sample enrichment analysis showed that B cells, T cells, and co-activation factors were significantly up-regulated in PPS. In addition, expression of CXCL13 and activation of CCL19/21-CCR7 axis were significantly increased, which indicated a PARPi-triggered “hot” immune-microenvironment. Moreover, immune-checkpoint TIGIT was significantly upregulated, paved the way for immunotherapy. Nevertheless, stromal remodeling, and tumor proliferation signatures were also up-regulated in PPS. In parallel, cancer-related pathways, PI3K/AKT, Epithelial-Mesenchymal transition, cell-cycle as well as G2M checkpoint were observed in the pathway enrichment analysis, which further verified the aforementioned findings. Conclusions: This small sample size study confirmed the complexity of the resistance mechanisms to PARPi, including BRCA reversion mutation, oncogene activation and bypass signaling activation. The activation of both positive and negative immune regulators in the tumor microenvironment shed light on future clinical management for patients with PARPi resistance.[Table: see text]
Recently periostin was associated with glioma angiogenesis and in a glioma mouse model it was shown that periostin is expressed by glioma stem cells, and its expression attracks tumor associated macrophages that promote malignant growth of glioma. In previous work on human gliomas we found periostin as specifically expressed in blood vessels of glioma as compared to other proliferating blood vessels. We now explored the cells that specifically express periostin at the protein and RNA level in human gliomas of various malignancy grades. I addition, in functional studies using gene silencing in cell cultures we studied the effects of periostin on angiogenesis and explored its regulators. We found that periostin is mainly expressed by (PDGFR-pos) pericytes and that these cells occasionally may display glioma stem cell characteristics. Co-culture experiments revealed that conditioned medium of the glioma cell line U87 increased the periostin expression by pericytes. Silencing of POSTN in pericytes in co-cultures with endothelial cells resulted in reduced formation, length and branching of vascular tubules. We conclude that periostin expression is crucial for glioma angiogenesis. In human glioma it is foremost expressed by pericytes and its expression is stimulated by glial tumor cells. The putative glioma stemness of pericytes needs further exploration. Clearly, the data open new avenues of anti-angiogenic strategies for glioma.
// Changbin Zhu 1, 6 , Dana A.M. Mustafa 1, * , Merle M. Krebber 5, * , Ihsan Chrifi 2 , Pieter J.M. Leenen 3 , Dirk J. Duncker 2 , Lennard Dekker 4 , Theo M. Luider 4 , Johan M. Kros 1, * and Caroline Cheng 2, 5, * 1 Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands 2 Division of Experimental Cardiology, Department of Cardiology, Erasmus Medical Center, Rotterdam, The Netherlands 3 Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands 4 Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands 5 Department of Nephrology and Hypertension, DIGD, University Medical Center Utrecht, Utrecht, The Netherlands 6 Department of Paediatric Neurosurgery, Shanghai Xin Hua Hospital/Shanghai Jiao Tong University School of Medicine, Shanghai, PR China * These authors contributed equally as corresponding authors to this work Correspondence to: Johan M. Kros, email: j.m.kros@erasmusmc.nl Keywords: tumor associated macrophages; glioma; CECR1; proteomics; immune response Abbreviations: TAM: tumor associated macrophage; CECR1: Cat Eye Syndrome Critical Region Protein 1; PMA: phorbol myristate acetate; MQ: macrophage; GBM: glioblastoma multiforme Received: August 24, 2017 Accepted: August 04, 2018 Published: September 11, 2018 ABSTRACT Introduction: Tumor associated macrophages (TAMs) promote tumor development, angiogenesis and distal metastasis. In previous studies, we showed that Cat Eye Syndrome Critical Region Protein 1 (CECR1) is expressed by M2-like TAMs in human glioma samples. CECR1 promoted M2 TAMs differentiation and affected glioma cell proliferation and migration. Here we investigated the proteomic profile of TAMs expressing CECR1 in absence or presence of glioma cells. Results: CECR1 siRNA transfection upregulated 67 proteins in THP-1-derived Macrophages (MQs). Pathway annotation mapped this set to 3 major pathways relevant for MQ function, including ‘MHC-I antigen presentation’, ‘phagosome maturation’ and ‘endocytosis’. Co-culture of siCECR1 THP-1-derived MQs with U87 glioma cells attenuated the changes observed on protein and mRNA level in response to MQ CECR1 silencing. SiCECR1 in U87 co-cultured MQs was associated with an IL-10 low , IL-12 high M1-like phenotype. In U87 co-culture conditions, SiCECR1 also downregulated S20 proteasome complex proteins PSMA5, PSMA7, PSMC6 and PSMD8. This protein profile was linked to a low proliferation rate of siCECR1 MQs. Overlap analysis identified S100A9 and PLAU as CECR1-related proteins that were significantly correlated with expression of CECR1 and macrophage lineage markers in three large public GBM datasets. Conclusion: This study reports the molecular pathways and key molecules that are mediated by CECR1 function in THP- 1-derived MQs and TAMs in glioma. Methods: PMA-treated THP-1 cells (MQs) were siRNA transfected for CECR1 in vitro , with or without stimulation of the primary glioma cell line U87. Lysates were analyzed by (nano)LC-MS. Significant altered protein levels were identified ( P < 0.05), followed by pathway annotation.
Background: Epidermal growth factor receptor (EGFR) 19 exon deletion (19del) has been observed to be associated with better outcomes in treatments with EGFR-TKIs than L858R mutation (L858R). It is of interest to explore whether the respective concomitant mutational profiles of 19del and L858R may explain their different sensitivities to EGFR-TKIs. Methods: We obtained next generation sequencing data to comprehensively determine the mutational profile of EGFR mutated patients from two cohort: our center (G1 cohort) and database cohort (G2 cohort). Mutation status of each gene and EGFR-TKI response information was retrieved. Gene Ontology (GO) biological process enrichment analyses of mutational profile were conducted. Findings: A total of 403 adenocarcinoma patients were recruited in G1 and 915 were included in G2 cohort. Similar prevalence of total concomitant mutations was observed in both G1 (19Del 34.30% vs. L858R 35.50%; p=0.803) and G2(19Del 73.4% vs. L858R 72.4%; p=0.735) cohort. The majority of mutations distribution between the two sub-types is similar in G1 and G2, however, CTNNB1(P=0.016), NTRK1(P=0.075) and U2AF1(P=0.075) were more frequent in L858R. Through logistic regression, we found total concomitant mutations (P=0.03), EGFR mutations (P=0.04) and T790M (P=0.02) independently affected the TKI response. In addition, 19Del showed higher ORR compared with L858R, regardless of concomitant mutations. Moreover, concomitant mutations in 19Del and L858R had various enrich signal pathway. Interpretations: Our study demonstrated that the prevalence of most concomitant mutations are similar between 19Del and L858R patients. Patients with L858R showed significantly lower response than those with 19del regardless of the co-mutation status.Funding Statement: The grant 2016YFC0905400 from the National Key R&D Program of China; China National Science Foundation (Grant No. 81871893 & No. 81501996); Key Project of Guangzhou Scientific Research Project (Grant No. 201804020030); High-level university construction project of Guangzhou medical university (Grant No. 20182737, 201721007, 201715907, 2017160107); National key R & D Program (Grant No. 2017YFC0907903 & 2017YFC0112704). Declaration of Interests: The authors state: "None declared."Ethics Approval Statement: The study protocol has been approved by the Research Ethics Committee of the First Affiliated Hospital of Guangzhou Medical university (S-201525). Written informed consent was obtained from all patients to permit genetic analysis of biological samples.
CCR5,as the member of CC receptor family,with its ligands being CCL3(MIP-1α),CCL4(MIP-1β) and CCL5(RANTES),is categorized as 7 trans-membrane domain G-protein coupled receptor.CCR5 is expressed in monocytes/macrophages as well as lymphocytes inducing chemotaxis and recruitment in inflammatory response and is an important co-receptor of human immunodeficiency virus(HIV)-1 virus,leading to the multifunction of CCR5 in progression of various kind of immunological diseases and invasion of HIV-1.Moreover,the expression of CCR5 on the surface of tumor cells and stromal cells contributes to mediating multiple biological behaviors of cancers such as proliferation and invasion.Advanced technologies also lead to the revealing of structure,function,signal transduction and roles of CCR5 in the progression of related diseases.
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