Rapid and reliable testing for clinically actionable EGFR mutations in non-small cell lung cancer using the IdyllaTM platform: a real-world two-center experience in Greece.

BACKGROUND: Limited information exists on epidermal growth factor receptor (EGFR) molecular epidemiology in Greece. Next-generation sequencing (NGS) is the recommended method for EGFR genotyping in NSCLC. The Idylla Biocartis platform is a fully automated system for actionable EGFR mutation detection. RESEARCH DESIGN AND METHODS: We describe the prevalence of EGFR mutations in NSCLC patients in two high-volume clinical centers in Greece and compare key methods used for their determination. Eight hundred and fifty-seven FFPE samples from NSCLC patients were tested for EGFR mutations at University of Crete (UoC; n = 324) and at Evangelismos Hospital, Athens (Evangelismos; n = 503). RESULTS: The prevalence of EGFR mutations was 11.1% in the whole cohort (11.5% in non-squamous). The detection rate was 11.0% by NGS, 9.8% by Sanger and 11.3% by Idylla for the whole cohort (12.0% in non-squamous). The agreement between Idylla and Sanger was 93.2%. A targetable EGFR mutation was detected in 10.0% using tissue NGS alone, and in 16.0% using concurrent Idylla ctEGFR testing. CONCLUSION: The frequency of EGFR mutations was as expected for a Caucasian population. The Idylla EGFR test performance is comparable to reference methods and with a shorter TAT. Adding a concurrent plasma Idylla test to tissue NGS testing increases the detection rate of EGFR mutations in NSCLC.

Molecular diagnostics for monitoring insecticide resistance in the western flower thrips Frankliniella occidentalis.

BACKGROUND: Insecticide resistance has emerged in various western flower thrips (WFT) populations across the world, threatening the efficiency of chemical control applications. Elucidation of insecticide resistance mechanisms at the molecular level provides markers for the development of diagnostics to monitor the trait and support evidence-based resistance management. RESULTS: TaqMan and Droplet Digital polymerase chain reaction (ddPCR) diagnostics were developed and validated, against Sanger sequencing, in individual and pooled WFT samples respectively, for the G275E mutation (nicotinic acetylcholine receptor α6 gene, nAChR α6) associated with resistance to nAChR allosteric modulators, site I (spinosyns); L1014F, T929I, T929C and T292V mutations (voltage-gated sodium channel gene, vgsc) linked with pyrethroid resistance; and I1017M (chitin synthase 1 gene, chs1) conferring resistance to growth inhibitors affecting CHS1 (benzoylureas). The detection limits of ddPCR assays for mutant allelic frequencies (MAF) were in the range of 0.1%-0.2%. The assays were applied in nine WFT field populations from Crete, Greece. The G275E (MAF = 29.66%-100.0%), T929I and T929V (combined MAF = 100%), L1014F (MAF = 11.01%-37.29%), and I1017M (MAF = 17.74%-51.07%) mutations were present in all populations. CONCLUSION: The molecular diagnostics panel that was developed in this study can facilitate the quick and sensitive resistance monitoring of WFT populations at the molecular level, to support evidence-based insecticide resistance management strategies. © 2022 Society of Chemical Industry.

Plasma-Based microRNA Expression Analysis in Advanced Stage NSCLC Patients Treated with Nivolumab.

Since circulating microRNAs (miRNAs) are involved in the modulation of the immune response, they are tested as liquid biopsy-based biomarkers in patients with NSCLC treated with immunotherapy. We analyzed the expression levels and examined the clinical significance of immunoregulatory miRNAs involved in immune checkpoint regulation (miR-34a, miR-200b, miR-200c), T-cell activity (miR-155), and the function of myeloid-derived suppressive cells (MDSCs) (miR-223) or regulatory T lymphocytes (Tregs) (miR-146a), in patients with advanced NSCLC (N = 69) treated with anti-PD-1 (Nivolumab) immunotherapy as 2nd or 3rd line of treatment therapy. Plasma levels of circulating miRNAs were analyzed by RT-qPCR before the initiation of immunotherapy. Expression of miR-34a, miR-146a, mir-200c, and miR-223 was found to be associated with response to immunotherapy. High miR-200c expression emerged as an independent prognostic factor for inferior overall survival in all patients with NSCLC (OS, HR: 2.243, 95% CI: 1.208-4.163; p = 0.010) and in patients with non-Squamous (non-SqCC) subtype (N = 38) (HR: 2.809, 95% CI: 1.116-7.074; p = 0.028). Low miR-34a expression independently predicted for shorter OS (HR: 3.189, 95% CI: 1.193-8.527; p = 0.021) in the non-SqCC subgroup. Our findings suggest that alterations in circulating miR-200c and miR-34a expression levels are associated with the response and outcome in patients with advanced NSCLC treated with anti-PD1 immunotherapy.

High Hereditary Transthyretin-Related Amyloidosis Prevalence in Crete: Genetic Heterogeneity and Distinct Phenotypes.

Background and Objectives: Our goal was to study hereditary transthyretin-related amyloidosis (hATTR) in Crete, Greece. Methods: We aimed at ascertaining all hATTR cases in Crete, an island of 0.62 million people. For this, we evaluated patients with polyneuropathy, autonomic involvement, cardiomyopathy, and/or ophthalmopathy suggestive of hATTR, who presented to the physicians of this study or were referred to them by other physicians. Genetic analyses were performed on all patients suspected of suffering from hATTR. We included in our observational longitudinal cohort study all individuals, residents of Crete, who, during the study period (1993-2019), were found to carry a pathogenic TTR variant. Results: Over the past 27 years, 30 individuals (15 female patients, 15 male patients), from 12 apparently unrelated families, were diagnosed with hATTR, whereas evaluation of their offspring identified 5 asymptomatic TTR pathogenic variant carriers. The most prevalent TTR variant detected was p.Val50Met, affecting 19 patients (11 female patients, 8 male patients) and causing a rather consistent phenotype characterized by predominant polyneuropathy of early adult onset (median age of symptom onset: 30 years; range: 18-37 years). Specifically, patients affected by the p.Val50Met TTR variant experienced progressive sensorimotor disturbances, involving mainly the lower extremities, associated with autonomic and/or gastrointestinal dysfunction. The second most frequent TTR variant was p.Val114Ala, found in 10 patients (4 female patients, 6 male patients) who were affected at an older age (median age of symptom onset: 70 years; range: 54-78 years). This variant caused a predominantly cardiomyopathic phenotype, manifested by congestive heart failure and associated with peripheral neuropathy, carpal tunnel syndrome, and/or autonomic involvement. In these patients, cardiac amyloid deposition was detected on 99m-technetium pyrophosphate scintigraphy and/or heart biopsy. The third TTR variant (p.Arg54Gly) was found in a 50-year-old male patient with ophthalmopathy due to vitreous opacities and positive family history for visual loss. As 22 patients were alive at the end of the study, we calculated the hATTR prevalence in Crete to be 35 cases per 1 million inhabitants. Discussion: Our study revealed that the prevalence of hATTR in Crete is one of the world's highest. Three different pathogenic TTR variants causing distinct clinical phenotypes were identified in this relatively small population pool.

Next-generation molecular diagnostics (TaqMan qPCR and ddPCR) for monitoring insecticide resistance in Bemisia tabaci.

BACKGROUND: Insecticide resistance has developed in several populations of the whitefly Bemisia tabaci worldwide and threatens to compromise the efficacy of chemical control. The molecular mechanisms underpinning resistance have been characterized and markers associated with the trait have been identified, allowing the development of diagnostics for individual insects. RESULTS: TaqMan and Droplet Digital PCR (ddPCR) assays were developed and validated, in individual and pooled whitefly samples, respectively, for the following target-site mutations: the acetylcholinesterase (ace1) F331W mutation conferring organophosphate-resistance; the voltage-gated sodium channel (vgsc) mutations L925I and T929V conferring pyrethroid-resistance; and the acetyl-CoA carboxylase (acc) A2083V mutation conferring ketoenol-resistance. The ddPCR's limit of detection (LoD) was <0.2% (i.e. detection of one heterozygote whitefly in a pool of 249 wild-type individuals). The assays were applied in 11 B. tabaci field populations from four locations in Crete, Greece. The F331W mutation was detected to be fixed or close to fixation in eight of 11 B. tabaci populations, and at lower frequency in the remaining ones. The pyrethroid-resistance mutations were detected at very high frequencies. The A2083V spiromesifen resistance mutation was detected in eight of 11 populations (frequencies = 6.16-89.56%). Spiromesifen phenotypic resistance monitoring showed that the populations tested had variable levels of resistance, ranging from full susceptibility to high resistance. A strong spiromesifen-resistance phenotype-genotype (A2083V) correlation (rs  = -0.839, P = 0.002) was observed confirming the ddPCR diagnostic value. CONCLUSION: The ddPCR diagnostics developed in this study are a valuable tool to support evidence-based rational use of insecticides and resistance management strategies. © 2022 Society of Chemical Industry.

TLR4 and pSTAT3 Expression on Circulating Tumor Cells (CTCs) and Immune Cells in the Peripheral Blood of Breast Cancer Patients: Prognostic Implications.

TLR4 and pSTAT3 are key players in cancer inflammation and immune evasion; however, their role in the peripheral blood (PB) is largely unexplored. Herein we evaluated their expression in the circulating tumor cells (CTCs) and peripheral-blood mononuclear cells (PBMCs) of patients with early (n = 99) and metastatic (n = 100) breast cancer (BC). PB samples obtained prior to adjuvant and first-line therapy, were immunofluorescently stained for Cytokeratins/TLR4/pSTAT3/DAPI and analyzed via Ariol microscopy. TLR4+ CTCs were detected in 50% and 68% of early and metastatic CTC-positive patients, respectively, and pSTAT3+ CTCs in 83% and 68%, respectively. In metastatic patients, CTC detection was associated with a high risk of death (HR: 1.764, p = 0.038), while TLR4+ CTCs correlated with a high risk of disease progression (HR: 1.964, p = 0.030). Regarding PBMCs, TLR4 expression prevailed in metastatic disease (p = 0.029), while pSTAT3 expression was more frequent in early disease (p = 0.014). In early BC, TLR4 expression on PBMCs independently predicted for high risk of relapse (HR: 3.549; p = 0.009), whereas in metastatic BC, TLR4+/pSTAT3- PBMCs independently predicted for high risk of death (HR: 2.925; p = 0.012). These results suggest that TLR4/pSTAT3 signaling on tumor- and immune-cell compartments in the PB could play a role in BC progression, and may hold independent prognostic implications for BC patients.

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy.

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically "lossy" liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.

Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae.

BACKGROUND: Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae (T. urticae), a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision-making for the rational use of acaricides. RESULTS: A suite of 12 TaqMan qPCR assays [G314D (GluCl1), G326E, I321T (GluCl3), G119S, F331W (Ace-1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1), G126S, S141F, P262T (cytb)], were validated against Sanger-sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI = 0.991-0.998), and no systematic, proportional, or random differences were detected. The achieved Limit of Detection (LoD) was 0.1% (detection of one mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases (e.g., etoxazole and abamectin resistance). Molecular diagnostics were able to capture incipient resistance that was otherwise missed by phenotypic bioassays. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi-resistant and susceptible populations. CONCLUSION: The highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. © 2021 Society of Chemical Industry.

May EPH/Ephrin Targeting Revolutionize Lung Cancer Treatment?

Lung cancer (LC) is the leading cause of cancer death in the United States. Erythropoietin-producing hepatocellular receptors (EPHs) comprise the largest receptor tyrosine kinases (RTKs) family in mammals. EPHs along with their ligands, EPH-family receptor-interacting proteins (ephrins), have been found to be either up- or downregulated in LC cells, hence exhibiting a defining role in LC carcinogenesis and tumor progression. In their capacity as membrane-bound molecules, EPHs/ephrins may represent feasible targets in the context of precision cancer treatment. In order to investigate available therapeutics targeting the EPH/ephrin system in LC, a literature review was conducted, using the MEDLINE, LIVIVO, and Google Scholar databases. EPHA2 is the most well-studied EPH/ephrin target in LC treatment. The targeting of EPHA2, EPHA3, EPHA5, EPHA7, EPHB4, EPHB6, ephrin-A1, ephrin-A2, ephrin-B2, and ephrin-B3 in LC cells or xenograft models not only directly correlates with a profound LC suppression but also enriches the effects of well-established therapeutic regimens. However, the sole clinical trial incorporating a NSCLC patient could not describe objective anti-cancer effects after anti-EPHA2 antibody administration. Collectively, EPHs/ephrins seem to represent promising treatment targets in LC. However, large clinical trials still need to be performed, with a view to examining the effects of EPH/ephrin targeting in the clinical setting.

Genetic cause of epilepsy in a Greek cohort of children and young adults with heterogeneous epilepsy syndromes.

We describe a cohort of 10 unrelated Greek patients (4 females, 6 males; median age 6.5 years, range 2-18 years) with heterogeneous epilepsy syndromes with a genetic basis. In these patients, causative genetic variants, including two novel ones, were identified in 9 known epilepsy-related genes through whole exome sequencing. A patient with glycine encephalopathy was a compound heterozygote for the p.Arg222Cys and the p.Ser77Leu AMT variant. A patient affected with Lafora disease carried the homozygous p.Arg171His EPM2A variant. A de novo heterozygous variant in the GABRG2 gene (p.Pro282Thr) was found in one patient and a pathogenic variant in the GRIN2B gene (p.Gly820Val) in another patient. Infantile-onset lactic acidosis with seizures was associated with the p.Arg446Ter PDHX gene variant in one patient. In two additional epilepsy patients, the p.Ala1662Val and the novel non-sense p.Phe1330Ter SCN1A gene variants were found. Finally, in 3 patients we observed a novel heterozygous missense variant in SCN2A (p.Ala1874Thr), a heterozygous splice site variant in SLC2A1 (c.517-2A>G), as a cause of Glut1 deficiency syndrome, and a pathogenic variant in STXBP1 (p.Arg292Leu), respectively. In half of our cases (patients with variants in the GRIN2B, SCN1A, SCN2A and SLC2A1 genes), a genetic cause with potential management implications was identified.

Highly sensitive droplet digital PCR-based diagnostics for the surveillance of malaria vector populations in low transmission and incipient resistance settings.

BACKGROUND: Sensitive monitoring of Plasmodium infective mosquitoes in low malaria transmission settings is of high priority for disease control. Early detection of insecticide resistance at low frequencies is also key for vector monitoring nowadays, when new insecticides are launched to control vector populations. RESEARCH DESIGN AND METHODS: An. gambiae mosquitoes with predetermined infection and resistance status were used to produce populations with various malaria infection rates and mutant allelic frequencies (MAFs) of target site insecticide resistance traits. Total RNA and gDNA were isolated and used in droplet Digital PCR (ddPCR) and Reverse Transcription (RT) ddPCR performed in the QX200 ddPCR System. RESULTS: We developed a novel ddPCR for detecting P. falciparum DNA in pooled mosquito head-thoraces with infective rate as low as 1.0%. A dissection-free RT-ddPCR assay for specific infective-stage detection was additionally developed and validated (accuracy = 100%) in mosquito pools with infective rates down to 1.0%. A novel ddPCR assay for insecticide resistant alleles, which was able to reliably quantify MAFs as low as 0.050% in pooled mosquito specimens, is also reported. CONCLUSIONS: We developed highly sensitive and efficient (RT-) ddPCR assays for contemporary operational needs that require monitoring of low malaria transmission and emerging insecticide resistance.

Genetic cause of heterogeneous inherited myopathies in a cohort of Greek patients.

Inherited muscle disorders are caused by pathogenic changes in numerous genes. Herein, we aimed to investigate the etiology of muscle disease in 24 consecutive Greek patients with myopathy suspected to be genetic in origin, based on clinical presentation and laboratory and electrophysiological findings and absence of known acquired causes of myopathy. Of these, 16 patients (8 females, median 24 years-old, range 7 to 67 years-old) were diagnosed by Whole Exome Sequencing as suffering from a specific type of inherited muscle disorder. Specifically, we have identified causative variants in 6 limb-girdle muscular dystrophy genes (6 patients; ANO5, CAPN3, DYSF, ISPD, LAMA2, SGCA), 3 metabolic myopathy genes (4 patients; CPT2, ETFDH, GAA), 1 congenital myotonia gene (1 patient; CLCN1), 1 mitochondrial myopathy gene (1 patient; MT-TE) and 3 other myopathy-associated genes (4 patients; CAV3, LMNA, MYOT). In 6 additional family members affected by myopathy, we reached genetic diagnosis following identification of a causative variant in an index patient. In our patients, genetic diagnosis ended a lengthy diagnostic process and, in the case of Multiple acyl-CoA dehydrogenase deficiency and Pompe's disease, it enabled specific treatment to be initiated. These results further expand the genotypic and phenotypic spectrum of inherited myopathies.

Detection of KRAS G12/G13 Mutations in Cell Free-DNA by Droplet Digital PCR, Offers Prognostic Information for Patients with Advanced Non-Small Cell Lung Cancer.

KRAS mutations are found in approximately one third of non-small cell lung cancer (NSCLC) patients. In this study, we aim to investigate whether KRAS G12/G13 mutant allele fraction (MAF) in cell-free DNA (cfDNA) can provide meaningful prognostic information in NSCLC. Multiplex droplet-digital PCR was used to quantitatively assess KRAS G12/G13 MAF in cfDNA from 114 pre-treated advanced disease NSCLC patients. In 14 patients, changes in KRAS G12/G13 MAF were longitudinally monitored during treatment. Plasma KRAS G12/G13 status was associated with poor patients' outcome in terms of progression-free survival (PFS) (p < 0.001) and overall survival (OS) (p < 0.001). In multivariate analysis, the detection of plasma KRAS mutations was an independent predictor of adverse PFS (HR = 3.12; p < 0.001) and OS (HR = 2.53; p = 0.002). KRAS G12/G13 MAF at first treatment evaluation (T1) was higher (p = 0.013) among patients experiencing progressive disease compared to those with disease control, and increased KRAS MAF at T1 was associated (p = 0.005) with shorter PFS. On the contrary, no association was observed between tissue KRAS mutation status and patients' prognosis. Our results show that ddPCR-based detection of KRAS G12/G13 mutations in plasma could serve as an independent biomarker of unfavorable prognosis in NSCLC patients. Changes in KRAS MAF can provide valuable information for monitoring patient outcome during treatment.

miR-181a overexpression predicts the poor treatment response and early-progression of serous ovarian cancer patients.

Ovarian cancer (OC) remains a leading cause of gynecological cancer-related death worldwide, characterized by poor 5-year survival. Molecular markers could serve as crucial tools of personalized prognosis and therapy. Herein, we present miR-181a as novel predictor of OC prognosis, using five independent OC cohorts. In particular, a screening (n = 81) and an institutionally independent validation (n = 100, OVCAD multicenter study) serous OC (SOC) cohorts were analyzed. Bagnoli et al (2016) OC179 (n = 124) to OC133 (n = 100) and TCGA (n = 489) served as external validation cohorts. Patients' survival and disease progression were assessed as clinical endpoint events. Bootstrap analysis was performed for internal validation and decision curve analysis was utilized to evaluate clinical benefit. miR-181a overexpression was unveiled as powerful and independent molecular predictor of patients' poor survival and higher risk for disease progression after debulking surgery and platinum-based chemotherapy. Analysis of the OVCAD institutionally independent cohort, as well as of Bagnoli et al. and TCGA external cohorts further confirmed the unfavorable prognostic nature of miR-181a overexpression in SOC. Strikingly, multivariate prognostic models incorporating miR-181a with established disease markers clearly improved patients' risk-stratification and offered superior clinical benefit in OC prognostication. Conclusively, miR-181a evaluation could augment prognostic accuracy and support precision medicine decisions in OC.

Whole exome sequencing establishes diagnosis of Charcot-Marie-Tooth 4J, 1C, and X1 subtypes.

BACKGROUND: Charcot-Marie-Tooth (CMT) hereditary polyneuropathies pose a diagnostic challenge. Our aim here is to describe CMT patients diagnosed by whole exome sequencing (WES) following years of fruitless testing. METHODS/RESULTS: Three patients with polyneuropathy suspected to be genetic in origin, but not harboring PMP22 gene deletion/duplication, were offered WES. The first patient, a 66-year-old man, had been suffering from progressive weakness and atrophies in the lower and upper extremities for 20 years. Due to ambiguous electrophysiological findings, immune therapies were administered to no avail. Twelve years after PMP22 deletion/duplication testing, WES revealed two pathogenic variants in the FIG4 gene (p.Ile41Thr and p.Phe598fs, respectively), as a cause of CMT 4J. The second patient, a 19-year-old man, had been suffering from hearing and gait impairment since at least his infancy, and recently presented with weakness and dystonia of the lower extremities. In this patient, WES identified the p.Leu122Val LITAF gene variant in heterozygous state, suggesting the diagnosis of CMT 1C, several years after initial genetic analyses. The third patient, a 44-year-old man, presented with progressive weakness and atrophies of the lower and upper extremities since the age of 17 years old. In this patient, WES identified the hemizygous p.Arg164Gln pathogenic variant in the GJB1 gene, establishing the diagnosis of CMT X1, 8 years after testing for PMP22 deletion/duplication. CONCLUSION: Novel diagnostic techniques, such as WES, offer the possibility to decipher the cause of CMT subtypes, ending the diagnostic Odyssey of the patients and sparing them from unnecessary and potentially harmful treatments.

Molecular markers related to immunosurveillance as predictive and monitoring tools in non-small cell lung cancer: recent accomplishments and future promises.

Introduction: The landscape of systemic treatment options for lung cancer has rapidly evolved with the emergence of immunomodulatory agents such as neutralizing antibodies targeting the programmed cell death protein 1 (PD-1) and its ligand (PD-L1). Another major breakthrough was the introduction of biomarkers, such as PD-L1 expression and tumor mutational burden (TMB), predicting response to immunotherapy. However, markers for monitoring treatment response are still lacking.Areas covered: PD-L1 and TMB represent static pre-treatment evaluations. Dynamic biomarkers are required, along with static ones, to accurately predict and monitor immunotherapy response and to discriminate between responders and non-responders early in the course of treatment. The tumor immune contexture offers potential candidates that can be tested through the liquid biopsy approach, such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes, microRNAs (miRNAs), circular RNAs (circRNAs), RNA splice variants, and immune cell subsets.Expert opinion: A holistic approach combining information from tissue at the time of diagnosis and serial liquid biopsy data could lead to a novel combinatorial biomarker panel with enhanced treatment monitoring potential. Incorporating information from additional parts of the tumor-host ecosystem, such as metabolic markers and the microbiome is expected to provide added value to this strategy.

miR-203 is an independent molecular predictor of prognosis and treatment outcome in ovarian cancer: a multi-institutional study.

Ovarian cancer (OC) accounts for the most gynecological cancer-related deaths in developed countries. Unfortunately, the lack of both evident early symptoms and effective asymptomatic population screening results in late diagnosis and inevitably poor prognosis. Hence, it is urgent to identify novel molecular markers to support personalized prognosis. In the present study, we have analyzed the clinical significance of miR-203 in OC using two institutionally independent cohorts. miR-203 levels were quantified in a screening (n = 125) and a validation cohort (n = 100, OVCAD multicenter study). Survival analysis was performed using progression and death as clinical endpoint events. Internal validation was conducted by bootstrap analysis, and decision curve analysis was used to evaluate the clinical benefit. Increased miR-203 levels in OC patients were correlated with unfavorable prognosis and higher risk for disease progression, independently of FIGO stage, tumor grade, residual tumor after surgery, chemotherapy response and age. The analysis of the institutionally independent validation cohort (OVCAD study) clearly confirmed the shorter survival outcome of the patients overexpressing miR-203. Additionally, integration of miR-203 levels with the established disease prognostic markers led to a superior stratification of OC patients that can ameliorate prognosis and benefit patient clinical management. In this regard, miR-203 expression constitutes a novel independent molecular marker to improve patients' prognosis in OC.

Localization of Human Glutamate Dehydrogenases Provides Insights into Their Metabolic Role and Their Involvement in Disease Processes.

Glutamate dehydrogenase (GDH) catalyzes the reversible deamination of L-glutamate to α-ketoglutarate and ammonia. In mammals, GDH contributes to important processes such as amino acid and carbohydrate metabolism, energy production, ammonia management, neurotransmitter recycling and insulin secretion. In humans, two isoforms of GDH are found, namely hGDH1 and hGDH2, with the former being ubiquitously expressed and the latter found mainly in brain, testis and kidney. These two iso-enzymes display highly divergent allosteric properties, especially concerning their basal activity, ADP activation and GTP inhibition. On the other hand, both enzymes are thought to predominantly localize in the mitochondrial matrix, even though alternative localizations have been proposed. To further study the subcellular localization of the two human iso-enzymes, we created HEK293 cell lines stably over-expressing hGDH1 and hGDH2. In these cell lines, immunofluorescence and enzymatic analyses verified the overexpression of both hGDH1 and hGDH2 iso-enzymes, whereas subcellular fractionation followed by immunoblotting showed their predominantly mitochondrial localization. Given that previous studies have only indirectly compared the subcellular localization of the two iso-enzymes, we co-expressed them tagged with different fluorescent dyes (green and red fluorescent protein for hGDH1 and hGDH2, respectively) and found them to co-localize. Despite the wealth of information related to the functional properties of hGDH1 and hGDH2 and the availability of the hGDH1 structure, there is still an ongoing debate concerning their metabolic role and their involvement in disease processes. Data on the localization of hGDHs, as the ones presented here, could contribute to better understanding of the function of these important human enzymes.

Human kallikrein-related peptidase 12 (KLK12) splice variants discriminate benign from cancerous breast tumors.

OBJECTIVES: As kallikrein-related peptidase 12 (KLK12) has been implicated in the cancer progression and alternative splicing plays significant role in this disease, the aim of this study was to examine the expression profile and the clinical impact of the KLK12 splice variants in breast cancer. DESIGN AND METHODS: Total RNA was isolated and reverse transcripted from 141 tissues. Afterwards, quantitative real-time PCR were conducted, followed by the performance of the comparative CT (2-ΔΔCT) method for relative quantification, whilst their correlation with the clinicopathological features of breast malignancies were assessed by statistical analysis. RESULTS: Both KLK12sv1/2 and KLK12sv3 showed higher expression in non-cancerous than in cancerous samples. KLKsv1/2 (P = 0.001) upregulated and KLK12sv3 (P < 0.001) downregulated in the malignant compared to the benign tumors and their discriminative ability was verified by ROC curve analysis. Moreover, KLK12sv3 was associated with grade (P = 0.012) and hormonal receptor status (P = 0.001). Furthermore, Kaplan-Meier and Cox regression analyses showed that patients with positive KLK12sv1/2 and KLK12sv3 levels presented a significantly longer disease-free survival (P = 0.014 and P = 0.013, respectively) and overall survival (P = 0.062 and P = 0.004, respectively). CONCLUSIONS: Our results demonstrate the discriminative value of KLK12sv1/2 and KLK12sv3 between benign and malignant breast tumors as well as their potential favorable prognostic significance in breast adenocarcinoma.