Independent Biomarker For Prognosis Research Articles

Identification of a bile acid and bile salt metabolism-related lncRNA signature for predicting prognosis and treatment response in hepatocellular carcinoma

Bile acids and salts have been shown to play a role in liver carcinogenesis through DNA damage, inflammation, and tumor proliferation. However, the correlation between bile acid metabolism and hepatocellular carcinoma (HCC) prognosis remains unclear. This study aimed to identify a predictive signature of bile acid and bile salt metabolism-related long non-coding RNAs (lncRNAs) for HCC prognosis and treatment response. The study used HCC RNA-sequencing data and corresponding clinical and prognostic data from The Cancer Genome Atlas. A prognostic model consisting of five bile acid and bile salt metabolism-related lncRNAs was developed and evaluated in a training set, a validation set and an external set. The model demonstrated good performance in predicting HCC prognosis and was shown to be an independent biomarker for prognosis. Additionally, our study revealed a significant association between the signature and immune cell infiltration, as well as its predictive value for therapeutic responses to both immunotherapy and chemotherapy. Furthermore, three LncRNAs (LUCAT1, AL031985.3 and AC015908.3) expression levels in our signature were validated through qRT-PCR in a cohort of 50 pairs of HCC patient tumor samples and corresponding adjacent non-tumor samples, along with 10 samples of normal liver tissue adjacent to benign lesions. These findings suggest that this novel bile acid and bile salt metabolism-related lncRNA signature can independently predict the prognosis of patients with HCC and may be utilized as a potential predictor of response to treatment in this setting.

SDHB immunohistochemistry for prognosis of pheochromocytoma and paraganglioma: A retrospective and prospective analysis.

Pheochromocytomas and paragangliomas (PCC/PGL) are rare neuroendocrine tumors and can secrete catecholamine. Previous studies have found that SDHB immunohistochemistry (IHC) can predict SDHB germline gene mutation, and SDHB mutation is closely associated with tumor progression and metastasis. This study aimed to clarify the potential effect of SDHB IHC as a predictive marker for tumor progression in PCC/PGL patients. We included PCC/PGL patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from 2002 to 2014 for retrospective analysis and discovered that SDHB (-) staining patients had poorer prognoses. Then we examined SDHB protein expression by IHC on all tumors in the prospective series, which was composed of patients from 2015 to 2020 in our center. In the retrospective series, the median follow-up was 167 months, and during follow-up, 14.4% (38/264) patients developed metastasis or recurrence, and 8.0% (22/274) patients died. Retrospective analysis revealed that 66.7% (6/9) of participants in the SDHB (-) group and 15.7% (40/255) of those in the SDHB (+) group developed progressive tumors (OR: 10.75, 95% CI: 2.72-52.60, P=0.001), and SDHB (-) was independently associated with poor outcomes after adjusting by other clinicopathological parameters (OR: 11.68, 95% CI: 2.58-64.45, P=0.002). SDHB (-) patients had shorter disease-free survival (DFS) and overall survival (OS) (P<0.001) and SDHB (-) was significantly associated with shorter median DFS (HR: 6.89, 95% CI: 2.41-19.70, P<0.001) in multivariate cox proportional hazard analysis. In the prospective series, the median follow-up was 28 months, 4.7% (10/213) patients developed metastasis or recurrence, and 0.5% (1/217) patient died. For the prospective analysis, 18.8% (3/16) of participants in the SDHB (-) group had progressive tumors compared with 3.6% (7/197) in the SDHB (+) group (RR: 5.28, 95% CI: 1.51-18.47, P=0.009), statistical significance remained (RR: 3.35, 95% CI: 1.20-9.38, P=0.021) after adjusting for other clinicopathological factors. Our findings demonstrated patients with SDHB (-) tumors had a higher possibility of poor outcomes, and SDHB IHC can be regarded as an independent biomarker of prognosis in PCC/PGL.

High Prolyl 4-Hydroxylase Subunit Alpha 3 Expression as an Independent Prognostic Biomarker and Correlated With Immune Infiltration in Gastric Cancer.

Background: Gastric cancer (GC) has a high mortality rate and is particularly prevalent in China. The extracellular matrix protein, prolyl 4-hydroxylase subunit alpha 3 (P4HA3), has been implicated in various cancers. We aimed to assess the diagnostic and prognostic value of P4HA3 in GC and investigate its correlation with immune cell infiltration. Methods: The present study used microarray data from the Cancer Genome Atlas (TCGA) to analyze the association of P4HA3 expression with clinicopathological features. Data from the Gene Expression Omnibus (GEO) were used for validation. Receiver operating characteristic (ROC) and Kaplan–Meier curves were constructed to determine the diagnostic and prognostic value of P4HA3 in GC. Univariate and multivariate regression analyses were performed to assess the impact of P4HA3 on overall survival (OS) rates. A protein–protein interaction (PPI) network was generated and functional enrichment evaluated. Single-sample gene set enrichment analysis (ssGSEA) was conducted to correlate P4HA3 expression with immune cell infiltration. The correlation between P4HA3 and immune check point genes was studied. Results: P4HA3 was over-expressed in GC, along with 15 other types of cancer, including breast invasive carcinoma and cholangiocarcinoma. P4HA3 showed high diagnostic and prognostic value in GC and was an independent prognostic factor. P4HA3, TNM (tumor, node, metastases) stage, pathological stage and age all correlated with OS rates. Genes related to P4HA3 were enriched in the lumen of the endoplasmic reticulum and included procollagen-proline 3-dioxygenase activity. P4HA3 expression correlated with numbers of macrophages, natural killer (NK) cells, immature dendritic cells (iDC), mast cells, eosinophils, effective memory T cells (Tem), T-helper 1 (Th1) cells and dendritic cells (DC). P4HA3 was positively correlated with hepatitis A virus cellular receptor 2 (HAVCR2) and programmed cell death 1 ligand 2 (PDCD1LG2). Conclusion: P4HA3 is a potential independent biomarker for prognosis of GC and may be an immunotherapy target in the treatment of GC.

TRIM24 Expression as an Independent Biomarker for Prognosis and Tumor Recurrence in HNSCC.

Background: Head and neck squamous cell carcinomas (HNSCCs) are among the most common cancers in humans worldwide and have a rather poor prognosis. TRIM24 has various intracellular functions and was identified in other cancer entities as a poor prognostic factor for patients. Methods: The expression of TRIM24 was evaluated by using immunohistochemistry. We used a large and representative cohort of 341 HNSCC patients. Data derived from immunohistochemistry evaluation was correlated with clinicopathological data from HNSCC patients. Results: The TRIM24 expression in HNSCC primary tumors is negatively correlated with the p16 status of the tumor tissues. Primary tumors of patients who developed a local recurrence were significantly more often positive for TRIM24. Kaplan–Meier analyses and Cox regression showed that patients with TRIM24 expressing tumors have significantly worse overall survival and progression-free survival and that TRIM24 expression is independent of other established risk factors. Conclusions: TRIM24 might be a new prognostic biomarker for the survival prognosis and early detection of local recurrences in HNSCC patients. It could be used for risk stratification of HNSCC patients and to identify those patients who are more prone to develop a local recurrence and therefore could profit from more frequent follow-up examinations.

Role of DTL in Hepatocellular Carcinoma and Its Impact on the Tumor Microenvironment.

BackgroundThe crucial role of DTL has been previously implicated in genomic stability; however, its prognostic value and its relation with tumor immunity in hepatocellular carcinoma (HCC) remain to be further explored.MethodsTranscriptional and mutational datasets as well as clinical information were retrieved from the GEO, ICGC, and TCGA databases. Differentially expressed genes (DEGs) were obtained from the comparison of DTLhigh and DTLlow expression groups of the TCGA-HCC cohort. Those genes were under KEGG and gene ontology (GO) analyses to decipher the influence of the DTL gene on the biological behavior of HCC tumor cells. The survival status and mutational characteristics of patients according to DTL levels were depicted and analyzed. The DTL overexpression in HCC and its impact on prognosis were further confirmed by a cohort of 114 HCC patients (validation cohort). The TIMER, GEPIA, and TISIDB databases were adopted to investigate the potential relations between DTL levels and the status of immune cells, as well as immune cell infiltrations.ResultsThe DTL gene is overexpressed in tumor tissues compared with distant non-malignant liver tissues, and DTL overexpression in HCC would enhance the HCC cells in the activities of cell cycle and division. HCC patients with high DTL expression have unfavorable clinical outcomes and harbor more somatic mutations than those with low DTL expression, and multivariate analysis also revealed that DTL overexpression could act as an independent biomarker for prognosis. Moreover, the DTL gene was positively linked to marker sets of infiltrating activated CD8+ and CD4+ T cells; however, these cells demonstrated to be functionally exhausted.ConclusionsPatients with a DTL overexpression phenotype in HCC have poorer prognosis than those in the DTLlow group due to the role of the DTL gene in the process of pro-cell proliferation, accompanied by the immunosuppressive microenvironment and T cell exhaustion.

Construction and validation of m6 A RNA methylation regulators associated prognostic model for gastrointestinal cancer.

N6‐methyladenosine (m6A) RNA methylation is correlated with carcinogenesis and dynamically possessed through the m6A RNA methylation regulators. This paper aimed to explore 13 m6A RNA methylation regulators' role in gastrointestinal cancer (GIC) and determine the risk model and prognosis value of m6A RNA methylation regulators in GIC. We used several bioinformatics methods to identify the differential expression of m6A RNA methylation regulators in GIC, constructed a prognostic model, and carried out functional enrichment analysis. Eleven of 13 m6A RNA methylation regulators were differentially expressed in different clinicopathological characteristics of GIC, and m6A RNA methylation regulators were nearly associated with GIC. We constructed a risk model based on five m6A RNA methylation regulators (METTL3, FTO, YTHDF1, ZC3H13, and WTAP); the risk score is an independent prognosis biomarker. Moreover, the five m6A RNA methylation regulators can also forecast the 1‐, 3‐ and 5‐year overall survival through a nomogram. Furthermore, four hallmarks of oxidative phosphorylation, glycolysis, fatty acid metabolism, and cholesterol homoeostasis gene sets were significantly enriched in GIC. m6A RNA methylation regulators were related to the malignant clinicopathological characteristics of GIC and may be used for prognostic stratification and development of therapeutic strategies.

MicroRNA-211-5p as a Biomarker in Early Detection of Uremic Vascular Calcification among Patients with End‐Stage Renal Disease

Vascular calcification (VC) is a serious pathological manifestation of vascular terminals and is an important pathological basis of cardiovascular diseases in patients with end-stage renal disease (ESRD). This study explored the clinical value of serum microRNA-211-5p (miR-211-5p) in VC in patients with ESRD, this study was designed. The relative expression of miR-211-5p in the serum of the calcified group and the non-calcified group was quantified by quantitative reverse transcription polymerase chain reaction. The clinical biochemical indexes and the expression differences in miR-211-5p expression in the serum of groups were compared by two independent samples t-test or nonparametric test or one-way analysis of variance test. The receiver operating characteristic (ROC), Kaplan-Meier, and Cox regression analysis were carried out to analyze the predictive value of serum miR-211-5p in patients with ESRD. The relative quantification of serum miR-211-5p was lessened in calcified group and gradually decreased with the progression of VC. Serum miR-211-5p had a high diagnostic accuracy in the diagnosis of VC progression in ESRD patients. Kaplan-Meier and Cox regression methods revealed that miR-211-5p might be an independent biomarker for prognosis of ESRD patients. MiR-211-5p is a potential diagnostic and prognostic marker of VC in patients with ESRD.

Neutrophil-to-Lymphocyte Ratio (NLR) Predicts PD-1 Inhibitor Survival in Patients with Metastatic Gastric Cancer.

Background and Aims Biomarkers for systemic inflammation have been introduced into clinical practice for risk-rating in cancer patients' treatment. This study is aimed at confirming the prognostic role of the neutrophil-to-lymphocyte ratio (NLR) as an effective biomarker for patients with metastatic gastric cancer (MGC) receiving anti-PD-1 agents. Method Patients with MGC who received anti-PD-1 treatment at the Chinese PLA General Hospital between January 2016 and November 2020 were reviewed. The study analyzed the association of NLR and overall survival (OS) or progression-free survival (PFS) and antitumor response rate with PD-1 inhibitors. Results 137 patients were included in the final analysis. The area under the curve value of NLR for 6-month OS was 0.71. The best cut-off value for NLR was 3.23. NLR < 3.23 was associated with longer OS (HR = 0.38, 95% CI, 0.26-0.57, p < 0.001) and PFS (HR = 0.42, 95% CI, 0.29-0.62, p < 0.001) in patients with MGC. No significant difference was observed in the objective response rate (ORR) (35.8% vs. 28.6%, p = 0.377) and disease control rate (DCR) (86.4% vs. 78.6%, p = 0.229) in the NLR < 3.23 group and in the NLR ≥ 3.23 group, respectively. Univariate analysis and multivariate analysis found that NLR was an independent prognosis biomarker for PFS and OS. Conclusions Pretreatment elevated NLR was significantly associated with inferior PFS and OS in patients with MGC who received anti-PD-1 inhibitors. Clinicians need to consider patients with elevated NLR for decisions on immunotherapy strategy.

RNA m1A Methyltransferase TRMT6 Predicts Poorer Prognosis and Promotes Malignant Behavior in Glioma.

Background: Glioma is the most prevalent central nervous system tumor in humans, and its prognosis remains unsatisfactory due to a lack of effective therapeutic targets. The ectopic expression of N1-methyladenosine (m1A) regulators is a key participant in tumorigenesis and progression. However, the m1A regulator expression status, prognostic value, and relationship with tumor clinical features in glioma remain unclear.Methods: Public datasets were used to analyze the mRNA and protein expression levels of m1A regulators. Kaplan–Meier and Cox regression analyses were performed to confirm the prognostic value of m1A regulators in glioma. Cellular experiments were conducted to verify the effect of TRMT6 on cell function. A comprehensive bioinformatics analysis was conducted to identify the potential molecular mechanisms regulated by TEMT6 in glioma.Results: We found that the dysregulation of m1A regulators was closely associated with tumorigenesis and progression in glioma. Furthermore, TRMT6 might be a powerful and independent biomarker for prognosis in glioma. Our study showed that inhibition of TRMT6 suppressed the proliferation, migration, and invasion of glioma cells. Mechanistically, TRMT6 may be involved in glioma progression by regulating cell cycle, PI3K-AKT, TGF-beta, MTORC1, NOTCH, and MYC pathways.Conclusions: Variation in m1A regulators was closely associated with malignant progression in glioma. Silencing TRMT6 suppressed the cell proliferation, migration, and invasion in glioma. m1A regulators, especially TRMT6, might play an essential role in the malignant progression of glioma.

Overexpression of P4HA1 associates with poor prognosis and promotes cell proliferation and metastasis of lung adenocarcinoma.

Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is the core active catalytic portion of prolyl 4-hydroxylase, and has contributed to tumorigenesis in several cancers. In this study, we identified that P4HA1 mRNA and protein are both up-regulated in non-small cell lung cancer (NSCLC). Besides, overexpressed P4HA1 is correlated with poor clinical outcomes and serve as an independent prognosis biomarker in lung adenocarcinoma (LUAD), but not lung squamous cell carcinoma (LUSC). In vitro studies, decreased P4HA1 significantly inhibits proliferation and cell cycle, by regulating cyclin-dependent kinases (CDKs), cyclins and CDK inhibitor (CKI). Moreover, via inhibiting epithelial-mesenchymal transition (EMT) and matrix metalloprotease (MMPs), dysregulation of P4HA1 could restrain the tumor cell invasion and metastasis of lung adenocarcinoma. In addition, we found that P4HA1 could enhance cell stemness and cisplatin-resistance in lung adenocarcinoma. In summary, P4HA1 plays a crucial role in the development of NSCLC and may provide a brand-new target for lung cancer treatment.

A glycolysis-related gene signature predicts prognosis of patients with esophageal adenocarcinoma.

Background: Esophageal adenocarcinoma (EAC) is a growing problem with a rapidly rising incidence and carries a poor prognosis. We aimed to develop a glycolysis-related gene signature to predict the prognostic outcome of patients with EAC.Results: Five genes (CLDN9, GFPT1, HMMR, RARS and STMN1) were correlated with prognosis of EAC patients. Patients were classified into high-risk and low-risk groups calculated by Cox regression analysis, based on the five gene signature risk score. The five-gene signature was an independent biomarker for prognosis and patients with low risk scores showed better prognosis. Nomogram incorporating the gene signature and clinical prognostic factors was effective in predicting the overall survival.Conclusion: An innovative identified glycolysis-related gene signature and an effective nomogram reliably predicted the prognosis of EAC patients.Methods: The Cancer Genome Atlas database was investigated for the gene expression profile of EAC patients. Glycolytic gene sets difference between EAC and normal tissues were identified via Gene set enrichment analysis (GSEA). Univariate and multivariate Cox analysis were utilized to construct a prognostic gene signature. The signature was evaluated by receiver operating characteristic curves and Kaplan–Meier curves. A prognosis model integrating clinical parameters with the gene signature was established with nomogram.

Transcriptome Analysis Identifies ALCAM Overexpression as a Prognosis Biomarker in Laryngeal Squamous Cell Carcinoma.

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most incident tumors in the world, especially in developing countries, such as Brazil. Different from other tumors, LSCC prognosis did not improve during the past four decades. Therefore, the objective of this study was to develop biomarkers that can predict LSCC patient’s prognosis. Results: Transcriptome analysis pointed out 287 overexpressed genes in LSCC in comparison to adjacent mucosa. Among these, a gene-pattern signature was created with 24 genes associated with prognosis. The Bayesian clustering of both Brazil and The Cancer Genome Atlas (TCGA) data pointed out clusters of samples possessing significative differences in the prognosis, and the expression panel of three genes (ALCAM, GBP6, and ME1) was capable to distinguish patients with worse prognosis with an accuracy of 97%. Survival analyses with TCGA data highlighted ALCAM gene expression as an independent prognostic factor for LSCC. This was further confirmed through immunohistochemistry, using a validation set of Brazilian patients. ALCAM expression was not associated with prognosis for other head and neck tumor sites. Conclusion: ALCAM overexpression seems to be an independent prognosis biomarker for LSCC patients.

Digital imaging-assisted quantification of H3K27me3 immunoexpression in luminal A/B-like, HER2-negative, invasive breast cancer predicts patient survival and risk of recurrence

BackgroundBreast cancer (BC) is a major health concern and better understanding of its biology might improve treatment decisions and patient outcomes. Histone3 Lysine27 tri-methylation (H3K27me3) is a post-translational histone modification frequently associated with altered gene expression. In BC patients, lower H3K27me3 expression has been associated with worse prognosis. We assessed H3K27me3 immunoexpression with digital imaging software assistance, in a cohort of luminal-like BC patients with long-term follow-up time and evaluated its association with clinically relevant endpoints and its clinical usefulness.MethodsH3K27me3 immunoexpression was assessed, by means of digital-imaging system, in archival tissue samples of 160 luminal A/B-like HER2-negative invasive BC, stages I-III. Survival analysis was performed using Kaplan-Meier and Cox regression. Cases were categorized as ‘low’ or ‘high’ expression based on cut-off defined by receiver operating characteristic (ROC) curve analysis.ResultsThe patient cohort showed a median age of 61-years, with a median follow-up time of 11.7 years. Low H3K27me3 expression (below 85% cut-off) was significantly associated with recurrence, both in univariable (HR = 1.99, 95%CI 1.066–3.724) and multivariable analysis when adjusting for grade and age (HR = 1.89, 95%CI 1.004–3.559). A trend for higher risk of death in low H3K27me3 expression BC was observed (p = 0.069), reaching statistical significance in younger patients (p = 0.021).ConclusionsH3K27me3 immunoexpression assessed by digital imaging scoring software is an independent prognosis biomarker in luminal-like BC patients and may assist in more individualized adjuvant treatment decisions, thus potentially reducing recurrences after curative-intent treatment, while sparing unnecessary toxicity.

A Five-microRNA Signature as Prognostic Biomarker in Colorectal Cancer by Bioinformatics Analysis.

Mounting evidence has demonstrated that a lot of miRNAs are overexpressed or downregulated in colorectal cancer (CRC) tissues and play a crucial role in tumorigenesis, invasion, and migration. The aim of our study was to screen new biomarkers related to CRC prognosis by bioinformatics analysis. By using the R language edgeR package for the differential analysis and standardization of miRNA expression profiles from The Cancer Genome Atlas (TCGA), 502 differentially expressed miRNAs (343 up-regulated, 159 down-regulated) were screened based on the cut-off criteria of p < 0.05 and |log2FC|>1, then all the patients (421) with differentially expressed miRNAs and complete survival time, status were then randomly divided into train group (212) and the test group (209). Eight miRNAs with p < 0.005 were revealed in univariate cox regression analysis of train group, then stepwise multivariate cox regression was applied for constituting a five-miRNA (hsa-miR-5091, hsa-miR-10b-3p, hsa-miR-9-5p, hsa-miR-187-3p, hsa-miR-32-5p) signature prognostic biomarkers with obviously different overall survival. Test group and entire group shown the same results utilizing the same prescient miRNA signature. The area under curve (AUC) of receiver operating characteristic (ROC) curve for predicting 5 years survival in train group, test group, and whole cohort were 0.79, 0.679, and 0.744, respectively, which demonstrated better predictive power of prognostic model. Furthermore, Univariate cox regression and multivariate cox regression considering other clinical factors displayed that the five-miRNA signature could serve as an independent prognostic factor. In order to predict the potential biological functions of five-miRNA signature, target genes of these five miRNAs were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway and Gene Ontology (GO) enrichment analysis. The top 10 hub genes (ESR1, ADCY9, MEF2C, NRXN1, ADCY5, FGF2, KITLG, GATA1, GRIA1, KAT2B) of target genes in protein protein interaction (PPI) network were screened by string database and Cytoscape 3.6.1 (plug-in cytoHubba). In addition, 19 of target genes were associated with survival prognosis. Taken together, the current study showed the model of five-miRNA signature could efficiently function as a novel and independent prognosis biomarker and therapeutic target for CRC patients.

1443P - Characterization of lung tumourspheres reveals cancer stem-like cells potential targets and prognostic markers in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) is the first cause of death cancer-related worldwide mainly due to high therapeutic resistance. This resistance is related to cancer stem-like cells (CSCs), for which the identification of targets and markers is still ongoing. MethodsPrimary cultures from 8 NSCLC patients were established as tumorspheres and as monolayers. CSCs properties were tested for both conditions in vitro and in vivo. The expression of 50 CSCs-related genes was assessed by RTqPCR and proteins of significantly overexpressed genes were examined by immunoblot and immunofluorescence. The prognostic role of these genes was analyzed in a cohort of 661 NSCLC patients from TCGA and validated in an independent cohort of 114 lung adenocarcinoma (ADC) patients. ResultsTumorspheres exhibited self-renewal, unlimited exponential growth, drug resistance, great invasion and differentiation capacities in vitro and higher tumorigenic potential than monolayers in vivo. 17 genes were significantly overexpressed in tumorspheres, being NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 the major contributors to distinguish them from adherent cells. Proteins encoded by these genes showed differential localization and expression patterns in ADC tumorspheres. The expression of CDKN1A, SNAI1 and ITGA6 was associated to prognosis, so a score was built based on their regression coefficients from a multivariate model. TCGA patients with high CSCs score show shorter OS in the entire cohort [37.7 vs. 60.4mo., p=0.001] and the ADC subcohort [36.6 vs. 53.5mo., p=0.003]. Multivariate analysis indicated that this score is an independent biomarker of prognosis for OS in the entire cohort from TCGA [HR: 1.498; 95% CI, 1.167-1.922; p=0.001] and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p=0.001]. The prognostic value is confirmed in an independent cohort of 114 lung adenocarcinoma patients OS (42.90 vs. NR mo, p=0.020). ConclusionsLung tumorspheres are a useful method for CSCs enrichment. Elevated expression of CDKN1A, SNAI1 and ITGA6 genes is associated with worse prognosis in NSCLC. Funded by CB16/12/00350 from CIBEROnc, PI12-02838, and PI15-00753 from ISCIII, Fundacion Arnal Planelles and Domingo Martínez. Legal entity responsible for the studyFundación de Investigación Hospital General de Valencia. FundingCIBEROnc, Instituto de Salud Carlos III; Fundacion Arnal Planelles and Domingo Martínez. DisclosureAll authors have declared no conflicts of interest.

Lung tumorspheres reveal cancer stem cell-like properties and a score with prognostic impact in resected non-small-cell lung cancer

The high resistance against current therapies found in non-small-cell lung cancer (NSCLC) has been associated to cancer stem-like cells (CSCs), a population for which the identification of targets and biomarkers is still under development. In this study, primary cultures from early-stage NSCLC patients were established, using sphere-forming assays for CSC enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using quantitative PCR, gene expression profiles were analyzed and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in a NSCLC cohort (N = 661) from The Cancer Genome Atlas. Based on a Cox regression analysis, CDKN1A, SNAI1, and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSC score. Kaplan–Meier survival analysis showed that patients with high CSC score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months (mo), p = 0.001] and the adenocarcinoma subcohort [36.6 vs. 53.5 mo, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167–1.922; p = 0.001] and the adenocarcinoma subcohort [HR: 1.869; 95% CI, 1.275–2.738; p = 0.001]. This score was also analyzed in an independent cohort of 114 adenocarcinoma patients, confirming its prognostic value [42.90 vs. not reached (NR) mo, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung adenocarcinoma patients and the basis for developing novel therapies. Further studies are required to identify suitable markers and targets for lung squamous cell carcinoma patients.

Is Visible Aminolevulinic Acid-Induced Fluorescence an Independent Biomarker for Prognosis in Histologically Confirmed (World Health Organization 2016) Low-Grade Gliomas?

BACKGROUNDApproximately 20% of low-grade gliomas (LGG) display visible protoporphyrin fluorescence during surgery after 5-aminolevulinic acid (5-ALA) administration.OBJECTIVETo determine if fluorescence represents a prognostic marker in LGG.METHODSSeventy-four consecutive patients with LGG (World Health Organization 2016) were operated on with 5-ALA. Fluorescent tissue was specifically biopsied. Tumor size, age, Karnofsky index, contrast-enhancement, fluorescence, and molecular factors (IDH1/IDH2-mutations, Ki67/MIB1 Index, 1p19q codeletions, ATRX, EGFR, p53 expression, and O6-methylguanine DNA methyltransferase promotor methylation), were related to progression-free survival (PFS), malignant transformation-free survival (MTFS) and overall survival (OS).RESULTSSixteen of seventy-four LGGs (21.6%) fluoresced. Fluorescence was partially related to weak enhancement on magnetic resonance imaging and increased (positron emission tomography)PET-FET uptake, but not to Karnofsky Performance Score, tumor size, or age. Regarding molecular markers, only EGFR expression differed marginally (fluorescing vs nonfluorescing: 19% vs 5%; P = .057). Median follow-up was 46.4 mo (95% confidence interval [CI]: 41.8-51.1). PFS, MTFS, and OS were shorter in fluorescing tumors (PFS: median 9.8 mo, 95% CI: 1.00-27.7 vs 45.8, 31.9-59.7, MTFS: 43.0 [27.5-58.5] vs 64.6 [57.7-71.5], median not reached, P = .015; OS: 51.6, [34.8-68.3] vs [68.2, 62.7-73.8], P = .002). IDH mutations significantly predicted PFS, MTFS, and OS. In multivariate analysis IDH status and fluorescence both independently predicted MTFS and OS. PFS was not independently predicted by fluorescence.CONCLUSIONThis is the first report investigating the role of ALA-induced fluorescence in histologically confirmed LGG. Fluorescence appeared to be a marker for inherent malignant transformation and OS, independently of known prognostic markers. Fluorescence in LGG might be taken into account when deciding on adjuvant therapies.

Chemokine Receptor CXCR3 Correlates with Decreased M2 Macrophage Infiltration and Favorable Prognosis in Gastric Cancer.

Aim The aim of this study was to explore the correlation of chemokine receptor CXCR3 with M2 macrophage infiltration, various clinicopathological features, and prognosis in patients diagnosed with gastric cancer (GC). Methods Expression of CXCR3 protein and M2 macrophage was evaluated in 156 GC patients and corresponding paracancerous tissues by immunohistochemical (IHC) analysis. Results In our study, 59 (37.82%) showed high expression of CXCR3 protein in 156 GC tissues. Expression of CXCR3 protein was significantly increased in tumor tissues compared with corresponding paracancerous tissues (P < 0.001). Overexpression of CXCR3 protein correlated with decreased M2 macrophage infiltration (P = 0.001). By analyzing the association between expression of CXCR3 protein and clinicopathological factors of GC patients, we found that high level of CXCR3 protein was significantly correlated with better differentiation (P =0.017), I/II TNM stage (P = 0.02), and smaller invasion depth (P = 0.003). Moreover, we found through Kaplan-Meier analysis and log-rank test that GC patients with high expression of CXCR3 protein and low M2 macrophage infiltration had better overall survival (OS) and low mortality rate (P < 0.001 and P = 0.024, respectively). The multivariate survival analysis showed that high expression of CXCR3 protein could serve as a favorable independent biomarker for prognosis in GC patients [hazard ratio (HR): 0.342 (0.204-0.571); P < 0.001]. Conclusion Our study indicates that overexpression of CXCR3 protein in GC is associated with decreased M2 macrophage infiltration and improved OS and thus can be further exploited as a biomarker in GC.

Discovery of a Novel Five Immune-Associated Fingerprints for Classification and Survival Prediction in Chronic Lymphocytic Leukemia

IntroductionCurrent staging methods do not accurately predict clinical outcome of patients with chronic lymphocytic leukemia (CLL) especially in the new era of immunotherapy. Recent studies suggested that immune-related gene signature predicted survival in glioblastoma, breast cancer and colorectal cancer. However, none related studies have been elucidated in CLL. Here, we hypothesized that risk-prediction model integrated with the immune signature could serve as an effective prognostic indicator in CLL. This study aimed to identify reliable and distinct immune-associated fingerprints for robust classification and survival prediction in patients with CLL.MethodsA total of 720 de novo CLL patients from multiple cohorts were enrolled with informed consents in the present study. LASSO Cox regression model was utilized to calculate immune-associated risk score (I-score) in R software. Principal Component Analysis (PCA) were performed to present the distribution of risk score. Moreover, the prognostic capability of the five immune-related fingerprints was demonstrated by PCR and ROC curve analysis with leave-one-out cross validation in the training set. Functional enrichment analyses of GO and KEGG in gene expression datasets were performed. Association between I-score and hallmark gene sets from the Molecular Signatures Database (MSigDB) were analyzed using GSEA software. Furthermore, preclinical experiments were conduct to examine the pathological mechanism of constitutive genes of I-score in CLL cell lines (MEC1, EHEB) and primary cells. Additionally, genomic regulatory network was displayed in Cytoscape software.ResultsIn the present study, we performed a comprehensive analysis to dissect the immune-associated fingerprints in CLL. A total of 305 clinical annotated CLL patients and 56 healthy donors with gene expression data were obtained from three independent cohorts. Two gene sets (immune system process, M13664 and immune response, M19817) were extracted from the MSigDB and combined to integrate the immune-related gene set containing 322 genes. Illustrated in the volcano plots, differentiated expressed genes of CLL cells comparing with normal B cells were calculated by limma test (|Log2Fold Change|>1, p<0.01; Figure 1A-C). Venn diagram was delineated to generate the specific CLL immune-associated gene expression panel, with 8 genes were identified (Figure 1D). PCA showed a different distribution pattern, confirming the enhanced immune phenotype in CLL.Then, we further investigate the association of the immuno-signature with clinical outcomes of CLL patients. By Lasso Cox regression analysis, the prognostic immune-related fingerprints were identified in the training set (Figure 2A). The risk score method was established: I-score = (-0.538)*CD3D expression+(-0.077)*CD83 expression+0.364*LAX1 expression+0.191*IL2RA expression+0.362*AIM2 expression, consisting of protective genes (CD3D, CD83) and risky genes (LAX1, IL2RA and AIM2; Figure 2B).Based on the median level of I-score as cut-off value, stratified high-risk patients were observed with significantly shorter overall survival compared with the low-risk group (Hazard Ratio, HR=5.493, p<0.001; Figure 3A). Univariate and multivariate cox regression analyses confirmed high I-score as an independent prognosis biomarker in CLL patients. To reduce disparity of diverse populations, we evaluated I-score in two independent centers of US and Germany with the same formula. The prognostic value of the immune fingerprints were corroborated in the internal validation series (HR=2.200, p=0.023; Figure 3B) and validation series (HR=1.769, p=0.004; Figure 3C). Intriguingly, we also observed the significant correlation between high I-score and 17p13 deletion (p=0.0316; Figure 3D), in accordance with patients' inferior outcome. Moreover, functional enrichment analyses of differentiated expressed genes stratified by I-score indicated that immune related BCR signaling pathway contributed to pathogenesis of CLL (Figure 4).ConclusionTo date, our study provides evidence for the first time that distinct immuno-related fingerprints predict survival in CLL. I-score is demonstrated as an efficient classification tool and robust method for prognosis evaluation, greatly facilitating risk stratification and individualized management of CLL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Validation of the Semiautomatic Quantification of 18F-Fluoride PET/CT Whole-Body Skeletal Tumor Burden.

Our purpose was to validate a semiautomatic quantification of the skeletal tumor burden on 18F-fluoride PET/CT using manual quantification as a reference. Methods: We quantified 51 18F-fluoride PET/CT examinations performed on female breast cancer patients. Clinical information (age; time of disease presentation; presence of visceral metastases; and time to death, progression, or a bone event) was recorded. The total volume of 18F-fluoride-avid skeletal metastases and the total activity of 18F-fluoride-avid metastases were calculated manually and semiautomatically. Results: Manual and semiautomatic metrics correlated strongly (P < 0.0001; 95% confidence interval, 0.9300-0.9769). On multivariable analysis, the semiautomatic measures of total activity for 18F-fluoride-avid metastasis correlated significantly with overall survival (P = 0.0001) and progression-free survival (P = 0.0006). Approximate times for calculating skeletal tumor burden (semiautomatic vs. manual) were, respectively, 30 s versus 321 s in patients with fewer than 5 metastases, 120 s versus 640 s in patients with 5-10 metastases, and 240 s versus 1207s in patients with more than 10 metastases. Conclusion: Semiautomatic quantification of whole-body 18F-fluoride PET/CT skeletal tumor burden can replace manual quantification in breast cancer patients and is a strong independent biomarker of prognosis.