4-gene Panel Research Articles

Genetic and clinical features of familial mediterranean fever (FMF) in a homogeneous cohort of patients from South-Eastern Italy

Familial Mediterranean Fever (FMF) is linked with the MEFV gene and is the commonest among monogenic autoinflammatory diseases, with high prevalence in the Mediterranean basin. Although the clinical presentation of FMF has a major role in diagnosis, genotype/phenotype correlations and the role of “benign” gene variants (as R202Q) appear highly variable and incompletely clear, making difficult to select the most effective strategy in the management of patients. Aim of the present study was to investigate the clinical presentation and the genetic background in a homogenous cohort of patients from Apulia (south eastern Italy). We investigated 217 patients with a clinical suspect of autoinflammatory diseases, who were characterized for the occurrence of specific symptoms and with next generation sequencing by a 4-gene panel including MEFV, MVK, NLRP3 and TNFRSF1A. A genetic change was identified in 122 (53.7%) patients, with 161 different MEFV variants recorded in 100 individuals, 10 variants in NLRP3, and 6 each in TNFRSF1A and MVK. The benign variant R202Q was largely prevalent (41.6% of all MEFV variants). When patients were selected according the number of pathogenic MEFV variants (0, 1, or 2 pathogenic variants), results failed to show significant links between the frequency of symptoms and the number of pathogenic variants. Only family history and Pras score (indicative for severity of disease) predicted the presence of pathogenic variants, as compared with carriers of variants considered of uncertain significance or benign. Fever >38 °C and arthralgias appeared more frequently in R202Q-positive patients than in non-R202Q carriers. These two subgroups showed comparable duration of fever, occurrence of myalgia, abdominal and chest pain, Pras, and IFFS scores. In conclusion, results confirm that FMF manifests in mild form in non-middle eastern patients. This possibility partly affects the reliability of clinical criteria/scores. Furthermore, the presence of the R202Q variant might not be completely neutral in selected groups of patients.

Prevalence of Undiagnosed Acute Hepatic Porphyria in Cyclic Vomiting Syndrome and Overlap in Clinical Symptoms.

Acute hepatic porphyria (AHP) presents with nausea and vomiting and can mimic cyclic vomiting syndrome (CVS). The prevalence of AHP in CVS and overlap in clinical symptomatology is not known. We thus sought to determine the prevalence of pathogenic variants for AHP and characterize symptom overlap between CVS and AHP. We conducted a cross-sectional study of 234 CVS patients using Rome criteria. Patients were eligible for AHP genetic testing if they had recurrent episodes of severe, diffuse abdominal pain with ≥ 2 of the following-peripheral nervous system (muscle weakness/aching, numbness, tingling), central nervous system (confusion, anxiety, seizures, hallucinations), autonomic nervous system (hyponatremia, tachycardia, hypertension, constipation) symptoms, red/brownish urine, or blistering skin lesions on sun-exposed areas. A family history of AHP or elevated urinary porphobilinogen (PBG)/aminolaevulinic acid (ALA) were also criteria for genetic testing and was performed using a 4-gene panel. Mean age was 38.7 ± 14.5years, 180 (76.9%) were female and 200 (85.5%) were Caucasian. During a CVS attack, 173 (92%) reported abdominal pain, 166 (87.2%) had peripheral nervous system, 164 (86.8%) had central nervous system and 173 (92) % had autonomic symptoms. Ninety-one eligible patients completed genetic testing. None were positive for AHP but two had variants of uncertain significance (VUS) in the HMBS gene. There is a high prevalence of non-gastrointestinal symptoms in CVS, like AHP, which is important for clinicians to recognize. AHP was not detected in this study and larger studies are warranted to ascertain its prevalence.

5-Hydroxymethylcytosines in circulating cell-free DNA and overall survival in patients with multiple myeloma.

8032 Background: The epigenetic mark 5-methylcytosines (5mC) have been associated with poor prognosis and survival in multiple myeloma (MM), but the prognostic role of 5-hydroxymethylcytosines (5hmC) as marks of tissue-specific enhancers generated from 5mC through active demethylation is unknown. We showed that 5hmC can be profiled in circulating cell-free DNA (cfDNA) and is associated with relapse/death in another lymphoproliferative disorder diffuse large B-cell lymphoma. To date, no study has investigated genome-wide 5hmC profiles in cfDNA for its prognostic significance in MM. Methods: A total of 354 newly diagnosed MM patients at the University of Chicago Medical Center were prospectively enrolled between 2010-2017. Blood samples were collected at the time of diagnosis. Patients were followed through 31 December 2020 (avg. follow-up = 77.8 mths). We collected baseline clinical, laboratory, and cytogenetic data from electronic medical records. Vital status was ascertained in 351 of the 354 patients (deaths = 73) using the National Death Index. We profiled genome-wide 5hmC in cfDNA using the 5hmC-Seal and next-generation sequencing. The 5hmC-Seal data were mapped to the human genome reference (hg19) and annotated to gene bodies. Overall survival (OS) was defined as time from diagnosis until death from any cause. We used Cox proportional hazards model and the elastic net regularization to identify genes with modified 5hmC levels that are associated with OS. Patients were randomly divided into a training set (n = 264) and testing set (n = 87). Results: The median age at diagnosis was 61.8 years and 47% (n = 165) were males. We used the differential 5hmC enrichment levels of a preliminary four-gene marker panel (i.e., YPEL1, VIPR2, PLAC8L1, and CYP2D6) to compute a weighted prognostic score (wp-score). In the training set (deaths = 55), MM patients with high wp-score had worse OS (Hazard Ratio [HR] = 2.2, 95% Confidence Interval [CI]: 1.3-3.9; p = 0.004). The same trend was observed in the testing set (deaths = 18) (HR = 3.5, 95% CI: 1.1-10.6; p = 0.02). The 5hmC-based wp-score remained significantly associated with OS after controlling for standard prognostic factors, suggesting that 5hmC-based wp-score for this 4-gene panel is an independent prognostic factor for MM. We also explored population-specific 5hmC and wp-score. We found that 5hmC profiles in cfDNA differ between Blacks (n = 117) and Whites (n = 234). In addition, 5hmC marker genes associated with OS differ between Blacks (13 genes) and Whites (20 genes). Conclusions: Our results suggest that 5hmC in cfDNA at the time of diagnosis correlate with OS in MM and the 5hmC marker genes associated with OS differ between Blacks and Whites. These findings suggest that a plasma-derived cfDNA 5hmC-modified gene panel holds promise as a noninvasive approach for predicting prognosis in MM and may be integrated in clinical practice to improve precision care.

Large-Scale Analysis Reveals Gene Signature for Survival Prediction in Primary Glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and common primary central nervous system tumour. Despite extensive therapy, GBM patients usually have poor prognosis with a median survival of 12–15 months. Novel molecular biomarkers that can improve survival prediction and help with treatment strategies are still urgently required. Here we aimed to robustly identify a gene signature panel for improved survival prediction in primary GBM patients. We identified 2166 differentially expressed genes (DEGs) using meta-analysis of microarray datasets comprising of 955 samples (biggest primary GBM cohort for such studies as per our knowledge) and 3368 DEGs from RNA-seq dataset with 165 samples. Based on the 1443 common DEGs, using univariate Cox and least absolute shrinkage and selection operator (LASSO) with multivariate Cox regression, we identified a survival associated 4-gene signature panel including IGFBP2, PTPRN, STEAP2 and SLC39A10 and thereafter established a risk score model that performed well in survival prediction. High-risk group patients had significantly poorer survival as compared with those in the low-risk group (AUC = 0.766 for 1-year prediction). Multivariate analysis demonstrated that predictive value of the 4-gene signature panel was independent of other clinical and pathological features and hence is a potential prognostic biomarker. More importantly, we validated this signature in three independent GBM cohorts to test its generality. In conclusion, our integrated analysis using meta-analysis approach maximizes the use of the available gene expression data and robustly identified a 4-gene panel for predicting survival in primary GBM.

A 4‐gene panel predicting the survival of patients with glioblastoma

To identify independently prognostic gene panel in patients with glioblastoma (GBM). The Cancer Genome Atlas (TCGA)-GBM was used as a training set and a test set. GSE13041 was used as a validation set. Survival associated differentially expression genes (DEGs), derived between GBM and normal brain tissue, was obtained using univariate Cox proportional hazards regression model and then was included in a least absolute shrinkage and selection operator penalized Cox proportional hazards regression model. Thus, a 4-gene prognostic panel was developed based on the risk score for each patient in that model. The prognostic role of the 4-gene panel was validated using univariate and multivariable Cox proportional hazards regression model. A total of 686 patients with GBMwere included in our study;724 DEGs was identified, 133 of which was significantly correlated with the overall survival (OS) of patients with GBM. A 4-gene panel including NMB, RTN1, GPC5, and epithelial membrane protein 3 (EMP3) was developed. Kaplan-Meier survival analysis suggested that patients in the 4-gene panel low risk group had significantly better OS than those in the 4-gene panel high risk group in the training set (hazard ratio [HR] = 0.3826;95% confidence interval [CI]: 0.2751-0.532;P < 0.0001), test set (HR = 0.718;95% CI: 0.5282-0.9759;P = 0.033) and the independent validation set (HR = 0.6898;95% CI: 0.4872-0.9766;P = 0.035). Both univariate and multivariable Cox proportional hazards regression analysis suggested that the 4-gene panel was independent prognostic factor for GBM in the training set. We developed and validated 4-gene panel that was independently correlated with the survival of patients with GBM.

Integrated diagnostic network construction reveals a 4-gene panel and 5 cancer hallmarks driving breast cancer heterogeneity

Breast cancer encompasses a group of heterogeneous diseases, each associated with distinct clinical implications. Dozens of molecular biomarkers capable of categorizing tumors into clinically relevant subgroups have been proposed which, though considerably contribute in precision medicine, complicate our understandings toward breast cancer subtyping and its clinical translation. To decipher the networking of markers with diagnostic roles on breast carcinomas, we constructed the diagnostic networks by incorporating 6 publically available gene expression datasets with protein interaction data retrieved from BioGRID on previously identified 1015 genes with breast cancer subtyping roles. The Greedy algorithm and mutual information were used to construct the integrated diagnostic network, resulting in 37 genes enclosing 43 interactions. Four genes, FAM134B, KIF2C, ALCAM, KIF1A, were identified having comparable subtyping efficacies with the initial 1015 genes evaluated by hierarchical clustering and cross validations that deploy support vector machine and k nearest neighbor algorithms. Pathway, Gene Ontology, and proliferation marker enrichment analyses collectively suggest 5 primary cancer hallmarks driving breast cancer differentiation, with those contributing to uncontrolled proliferation being the most prominent. Our results propose a 37-gene integrated diagnostic network implicating 5 cancer hallmarks that drives breast cancer heterogeneity and, in particular, a 4-gene panel with clinical diagnostic translation potential.

Integrated analysis of promoter methylation and expression of telomere related genes in breast cancer.

Telomeres at the ends of eukaryotic chromosomes play a critical role in tumorgenesis. Using microfluidic PCR and next-generation bisulfite sequencing technology, we investigated the promoter methylation of 29 telomere related genes in paired tumor and normal tissues from 184 breast cancer patients. The expression of significantly differentially methylated genes was quantified using qPCR method.We observed that the average methylation level of the 29 telomere related genes was significant higher in tumor than that in normal tissues (P = 4.30E-21). A total of 4 genes (RAD50, RTEL, TERC and TRF1) showed significant hyper-methylation in breast tumor tissues. RAD51D showed significant methylation difference among the four breast cancer subtypes. The methylation of TERC showed significant association with ER status of breast cancer. The expression profiles of the 4 hyper-methylated genes showed significantly reduced expression in tumor tissues. The integration analysis of methylation and expression of these 4 genes showed a good performance in breast cancer prediction (AUC = 0.947).Our results revealed the methylation pattern of telomere related genes in breast cancer and suggested a novel 4-gene panel might be a valuable biomarker for breast cancer diagnosis.

Abstract LB-155: Identification of a DNA methylation signature in liquid biopsy for early non-small cell lung cancer (NSCLC) diagnosis

Abstract Introduction: Lung cancer is the leading cause worldwide, mainly due to late diagnosis. The aim of this work was to identify a panel of epigenetic biomarkers for improving early diagnosis of lung cancer patients using minimally and non-invasive biological fluids. Patients and Methods: DNA hypermethylated biomarkers were identified performing a Genome-wide DNA methylation analysis (Infinium 450K array) in Non-small cell lung cancer (NSCLC) primary tumors from two different public databases (Discovery cohorts): CURELUNG FP7 Consortium (237 stage I NSCLC, 25 non-tumoral lung samples) and The Cancer Genome Atlas (TCGA; 350 stage I NSCLC, 62 non-tumoral lung samples). DNA methylation levels of selected candidates were analyzed by pyrosequencing in non- or minimally invasive samples from three independent cohorts of stage I NSCLC patients and non-tumoral controls (Validation cohorts): bronchoalveolar aspirates (82 NSCLC; 29 controls), bronchoalveolar lavages (51 NSCLC; 29 controls) and sputum (72 NSCLC; 26 controls). Combined Receiver Operating Characteristic (ROC) curve was obtained to evaluate the diagnostic utility of the epigenetic signature. Results: We identified a panel of 4 cancer-specific genes (BCAT1, CDO1, TRIM58 and ZNF177) with CpG island hypermethylation-associated silencing in early stage NSCLC primary tumors. All these genes presented significantly higher mean levels of%methylation (M) in NSCLC primary tumors respect to non-tumoral controls: BCAT1 (NSCLC: M&amp;gt;50%; Controls: M&amp;lt;20%), CDO1 (NSCLC: M&amp;gt;40%; Controls: M&amp;lt;10%), TRIM58 (NSCLC: M&amp;gt;50%; Controls: M&amp;lt;20%) and ZNF177 (NSCLC: M&amp;gt;40%; Controls : M&amp;lt;20%). Importantly, the diagnostic utility of the combination of this 4-gene panel signature was validated in liquid biopsy, showing a very high diagnostic accuracy with areas under the ROC curve (AUC) close to the maximum value: bronchoalveolar aspirates (AUC = 0.91; 95% CI [0.83, 0.98]; p &amp;lt; 0.001), bronchoalveolar lavages (AUC = 0.85; 95% CI [0.78, 0.93]; p &amp;lt; 0.001) and sputum (AUC = 0.93; 95% CI [0.86, 1.0]; p &amp;lt; 0.001). Conclusions: The herein identified DNA methylation signature could improve, in combination with current diagnostic protocols, the early diagnosis and outcome of NSCLC patients. The high diagnostic accuracy of this signature obtained in liquid biopsy offers a minimally invasive and easy accessible tool for early lung cancer diagnosis. Citation Format: A Diaz-Lagares, J Mendez-Gonzalez, D Hervas, M Saigi, MJ Pajares, D Garcia, AB Crujeiras, R Lopez-Lopez, R Pio, LM Montuenga, JJ Zulueta, E Nadal, A Rosell, M Esteller, J Sandoval. Identification of a DNA methylation signature in liquid biopsy for early non-small cell lung cancer (NSCLC) diagnosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-155.

MP02-07 NOVEL URINE MARKERS FOR DIAGNOSING AND MONITORING NON-INDOLENT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2016MP02-07 NOVEL URINE MARKERS FOR DIAGNOSING AND MONITORING NON-INDOLENT PROSTATE CANCER Daniella B. Frias, Ilsa L. Coleman, John S. Banerji, Khanh Pham, Claudio Jeldres, Roman Gulati, Jing Xia, Scott Tomlins, Christopher Porter, and Peter S. Nelson Daniella B. FriasDaniella B. Frias More articles by this author , Ilsa L. ColemanIlsa L. Coleman More articles by this author , John S. BanerjiJohn S. Banerji More articles by this author , Khanh PhamKhanh Pham More articles by this author , Claudio JeldresClaudio Jeldres More articles by this author , Roman GulatiRoman Gulati More articles by this author , Jing XiaJing Xia More articles by this author , Scott TomlinsScott Tomlins More articles by this author , Christopher PorterChristopher Porter More articles by this author , and Peter S. NelsonPeter S. Nelson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1879AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) is the most common solid tumor in men. Active surveillance (AS) is an acceptable alternative to immediate treatment for low-risk, Gleason pattern 3 (GP3) PCa. Furthermore, recent reports from Klotz et al in Toronto have demonstrated the outcomes of both intermediate risk, Gleason sum 7, and low risk Gleason 6 PCa in men on AS. Importantly men with intermediate risk fare significantly worse on AS. AS often entails significant risk of under-grading the tumor and repeated biopsies. We hypothesized that transcripts associated with high Gleason grade cancers are quantifiable in urine samples and reflect the presence of higher-grade non-indolent tumors. Our objective was to develop a qPCR-urine-based assay for the detection of occult high-grade cancer GP4 Vs. GP3 disease, in urine samples from men diagnosed with PCa. METHODS We performed a prospective IRB approved evaluation of 53 men undergoing radical prostatectomy (RP) for clinically localized disease. In the operating room prior to RP men underwent a digital rectal exam (DRE) followed by collection of urine by catheterization. Final pathology reviewed by a single GU pathologist was used as the gold standard for determination of final histological Grade. We had previously identified 4 consistently up-regulated GP4 transcripts (RELN, GRIN3A, RGS5 and LRNN1) Quantitative PCR (qPCR) was performed for the GP4 markers and normalized to KLK3 and RPL13A. By comparing transcriptional profiles of GP3 and GP4 cancers, we identified transcripts differentially expressed between these histologies. To evaluate the ability of the GP4 markers to discriminate between low and high-grade tumors, univariate and multivariate logistic regression analyses were performed and the overall significance determined using likelihood ratio tests. RESULTS Each gene was an independent predictor of high-grade (Gleason score =4+3 vs. =3+4) prostate cancer (p<0.05 for each gene) with area under receiver operating characteristic curves (AUC) of 0.786, 0.770, 0.794, and 0.784. When tested as a panel, the 4 genes were significantly associated with high-grade cancer (Primary pattern Gleason 4 disease) based on the overall likelihood ratio test (p=0.002). Furthermore, the 4-gene panel outperformed each gene alone, with an AUC of 0.841. CONCLUSIONS A novel 4-gene panel assayed by qPCR in men undergoing RP appears to be capable of discriminating clinically relevant high-grade (GP4 + GP3) tumors from low-grade (GP3+ GP4 or less) tumors using urine sediments. Further studies are needed to confirm these preliminary findings. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e11-e12 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Daniella B. Frias More articles by this author Ilsa L. Coleman More articles by this author John S. Banerji More articles by this author Khanh Pham More articles by this author Claudio Jeldres More articles by this author Roman Gulati More articles by this author Jing Xia More articles by this author Scott Tomlins More articles by this author Christopher Porter More articles by this author Peter S. Nelson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Utilization of Molecular Testing in Thyroid Cytology

While benign and malignant diagnoses rendered by fine-needle aspiration cytology (FNAC) are quite accurate, there remains a group of indeterminate lesions that are diagnostically challenging and difficult to preoperatively assign to a specific biologic behavior. These indeterminate lesions make up approximately 30% of thyroid FNAC and include atypia/follicular lesion of undetermined significance (AUS/FLUS), suggestive of follicular neoplasm (SFN), and suggestive of malignancy. With increasing knowledge about molecular mechanisms used in cancer, and lack of definitive cytologic characterization of indeterminate lesions, utilization of molecular techniques has become an attractive option to clinicians. The best available commercial tests today are miRInform (Asuragen) and Afirma (Veracyte), a 4-gene panel and gene expression classifier that are used to confirm or exclude the diagnosis of malignancy, respectively. Afirma test performs best in AUS/FLUS cases, but should be considered only if surgery is an option and only after repeat FNAC diagnosis of AUS/FLUS or SFN. Either or both molecular tests may be utilized in SFN cases, to confirm a benign or malignant clinical impression, and/or extent of surgery. Both of these molecular panels, however, are associated with significant false-positive and false-negative results. We believe that molecular testing can contribute to patient management, as an ancillary tool, in selected AUS/FLUS and SFN cases, but has no significant benefit to patients with lesions suggestive of malignancy. Molecular studies should not be ordered indiscriminately as a reflex test, and their results must not outweigh clinical judgment.

A 4-gene panel as a marker at chromosome 8q in Asian gastric cancer patients

A widely held viewpoint is that the use of multiple markers, combined in some type of algorithm, will be necessary to provide high enough discrimination between diseased cases and non-diseased. We applied stepwise logistic regression analysis to identify the best combination of the 32 biomarkers at chromosome 8q on an independent public microarray test set of 80 paired gastric samples. A combination of SULF1, INTS8, ATP6V1C1, and GPR172A was identified with a prediction accuracy of 98.0% for discriminating carcinomas from adjacent noncancerous tissues in our previous 25 paired samples. Interestingly, the overexpression of SULF1 was associated with tumor invasion and metastasis. Function prediction analysis revealed that the 4-marker panel was mainly associated with acidification of intracellular compartments. Taken together, we found a 4-gene panel that accurately discriminated gastric carcinomas from adjacent noncancerous tissues and these results had potential clinical significance in the early diagnosis and targeted treatment of gastric cancer.