Molecular Genomic Assessment Using a Blood-based mRNA Signature (NETest) is Cost-effective and Predicts Neuroendocrine Tumor Recurrence With 94% Accuracy.

INTRODUCTION: Identification of residual disease after neuroendocrine tumor (NET) resection is critical for management. Post-surgery imaging is insensitive, expensive, and current biomarkers ineffective. We evaluated whether the NETest, a multigene liquid biopsy blood biomarker, correlated with surgical resection and could predict recurrence. METHODS: Multicenter evaluation of NET resections over 24 months (n = 103): 47 pancreas, 26 small bowel, 26 lung, 2 appendix, 1 duodenum, 1 stomach. Surgery: R0 (83), R1/R2 (20). One millilitre of blood was collected at D0 and posroperative day (POD) 30. Transcript quantification by polymerase chain reaction (normal: ≤20), CgA by NEOLISA (normal ≤108 ng/mL). Standard-of-care (SoC) follow-up costs were calculated and compared to POD30 NETest-stratification approach. Analyses: Wilcoxon-paired test, Chi-square test. D BIOMARKERS: NETest: 103 of 103 (100%)-positive, whereas 23 of 103 (22%) were CgA-positive (Chi-square = 78, P < 0.0001).In the R0 group, the NETest decreased 59 ± 28 to 26 ± 23 (P < 0.0001); 36% (30/83) remained elevated. No significant decrease was evident for CgA. In the R1/R2 group the NETest decreased but 100% remained elevated. CgA levels did not decrease.An elevated POD30 NETest was present in R0 and 25 (83%) developed radiological recurrences. Normal score R0 s (n = 53) did not develop recurrence (Chi-square = 56, P < 0.0001). Recurrence prediction was 94% accurate with the NETest. COST EVALUATION: Using the NETest to stratify postoperative imaging resulted in a cost-savings of 42%. CONCLUSION: NETest diagnosis is more accurate than CgA (100% vs 22%). Surgery significantly decreased NETest. An elevated POD30 NETest predicted recurrence with 94% accuracy and post-surgical POD30 NETest follow-up stratification decreased costs by 42%. CgA had no surgical utility. Further studies would define the accuracy and cost-effectiveness of the NETest in the detection of postoperative recurrent disease.

High-fat meal increases peripheral blood mononuclear cell pro-inflammatory cytokine expression in African-American women.

African-American (AA) women have elevated predominance of inflammatory diseases concurrent with local inflammation resulting in compromised metabolic function. The purpose of the study was 2-fold: 1) to examine the gene and protein expression of pro- and anti-inflammatory cytokine secretion by peripheral blood mononuclear cells (PBMC) obtained from AA and Caucasian-American (CA) women in response to an acute high-fat meal; and 2) to explore the influence of race (AA vs. CA) on PBMC reactivity. Ten AA and 11 CA women consumed a high-fat meal with baseline and 4 h postprandial venous blood draws. PBMCs were incubated for 3 h then messenger RNA expression and supernatant protein concentration was used to examine inflammatory profiles. All women had a postprandial increase in interleukin (IL)-8 gene expression, IL-8 protein concentration, and tumor necrosis factor alpha (TNF-α) protein concentration (P < 0.05). AA women had a postprandial increase in IL-6, IL-8, and TNF-α protein concentration (P < 0.05). AA women had higher postprandial IL-1ß protein concentration and IL-8 gene expression compared with CA women (P < 0.05). Our data uncovers the specific impact of race and time on pro-inflammatory PBMC (IL-1ß, IL-6, IL-8, and TNF-α) expression profiles in response to an acute high-fat meal challenge. Novelty: African Americans have higher predominance of inflammatory disease. We explored the potential race impact on peripheral blood mononuclear cell reactivity in response to a meal. A pro-inflammatory response to an acute high-fat meal with race impact was observed possibly contributing to health disparities impacting African-American women.

Assessment of lncRNA GAS5, lncRNA HEIH, lncRNA BISPR and its mRNA BST2 as serum innovative non-invasive biomarkers: Recent insights into Egyptian patients with hepatitis C virus type 4.

BACKGROUND: Hepatitis C virus (HCV) infection and its consequent complications are undeniably a public health burden worldwide, particularly in Egypt. Emerging evidence suggests that many lncRNAs have relevant roles in viral infections and antiviral responses. AIM: To investigate the expression profiles of circulating lncRNAGAS5, lncRNAHEIH, lncRNABISPR and mRNABST2 in naïve, treated and relapsed HCV Egyptian patients, to elucidate relation to HCV infection and their efficacy as innovative biomarkers for the diagnosis and prognosis of HCV GT4. METHODS: One hundred and thirty HCV-infected Egyptian patients and 20 healthy controls were included in this study. Serum lncRNAs and mRNABST2 were measured using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our results indicated that serum lncRNAGAS5 and LncRNABISPR were upregulated, whereas mRNA BST2 and LncRNA HEIH were downregulated in naïve patients. In contrast, HCV patients treated with sofosbuvir and simeprevir; with sofosbuvir and daclatasvir; or with sofosbuvir, daclatasvir and ribavirin exhibited lower levels of lncRNAGAS5 and lncRNABISPR with higher mRNABST2 compared to naïve patients. Notably, patients relapsed from sofosbuvir and simeprevir showed higher levels of these lncRNAs with lower mRNABST2 compared to treated patients. LncRNAGAS5 and lncRNABISPR were positively correlated with viral load and ALT at P < 0.001, whereas mRNABST2 was negatively correlated with viral load at P < 0.001 and ALT at P < 0.05. Interestingly, a significant positive correlation between lncRNA HEIH and AFP was observed at P < 0.001. CONCLUSION: Differential expression of these RNAs suggests their involvement in HCV pathogenesis or antiviral response and highlights their promising roles in diagnosis and prognosis of HCV.

Gene expression profiling of whole blood: A comparative assessment of RNA-stabilizing collection methods.

Peripheral Blood gene expression is widely used in the discovery of biomarkers and development of therapeutics. Recently, a spate of commercial blood collection and preservation systems have been introduced with proprietary variations that may differentially impact the transcriptomic profiles. Comparative analysis of these collection platforms will help optimize protocols to detect, identify, and reproducibly validate true biological variance among subjects. In the current study, we tested two recently introduced whole blood collection methods, RNAgard® and PAXgene® RNA, in addition to the traditional method of peripheral blood mononuclear cells (PBMCs) separated from whole blood and preserved in Trizol reagent. Study results revealed striking differences in the transcriptomic profiles from the three different methods that imply ex vivo changes in gene expression occurred during the blood collection, preservation, and mRNA extraction processes. When comparing the ability of the three preservation methods to accurately capture individuals' expression differences, RNAgard® outperformed PAXgene® RNA, and both showed better individual separation of transcriptomic profiles than PBMCs. Hence, our study recommends using a single blood collection platform, and strongly cautions against combining methods during the course of a defined study.

Assessment of Apoptosis Pathway in Peripheral Blood of Autistic Patients.

Autism spectrum disorder (ASD) includes a number of severe neurodevelopmental disorders known by defects in social interaction, impaired verbal and non-verbal interactions, and stereotypic activities and limited interests. Dysregulation of apoptotic pathways have been demonstrated in brain tissues of affected individuals. In the present study, we evaluated expression levels of apoptosis-related genes and miRNAs in peripheral blood of ASD patients compared with healthy subjects. Transcript levels of BCL2, CASP8, and hsa-29c-3p were significantly lower in total ASD patients compared with total normal children (P values = 0.003, 0.002, and 0.01 respectively). When sex of study participants was considered in the analysis, the difference in transcript levels of these genes was significant only in male subjects. Peripheral expression of BCL2 and hsa-29c-3p had 100% sensitivity 92% specificity in ASD diagnosis. The diagnostic power of combination of transcript levels of these genes was estimated to be 78% based on the calculated AUC value. The present study provides evidences for dysregulation of apoptotic pathways in peripheral blood of ASD patients and suggests certain apoptosis-related genes as biomarkers in this regard.

Development and validation of a peripheral blood mRNA assay for the assessment of antibody-mediated kidney allograft rejection: A multicentre, prospective study.

BACKGROUND: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available. METHODS: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661. FINDINGS: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p < 0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection. INTERPRETATION: We identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection. FUNDING: The Seventh Framework Programme (FP7) of the European Commission.

Androgen receptor mRNA analysis from whole blood: a low-cost strategy for detection of androgen receptor gene splicing defects.

Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) gene and is the most common aetiology of 46,XY disorders of sex development. Allelic variants in the AR gene are found in 90% of complete AIS (CAIS), but in only 28% to 50% of cases of partial AIS. Even a single nucleic acid change can disrupt splicing sites or splicing regulatory sequences, resulting in inadequate exon and intron recognition, ultimately leading to an aberrant transcript. Therefore, we tested the feasibility of conducting AR cDNA analysis from whole blood and from gonadal tissue in a patient with CAIS due to AR synonymous mutation (c.1530C > T, p.Ser510Ser; NM_000044.3), which led to an aberrant splicing site causing deletion of 92 nucleotides resulting in a very short transcript. AR cDNA sequencing was similar in the whole blood and in the gonadal tissue, with similar evidence of a consequent altered AR transcript. We propose that analysis of AR RNA extracted from whole blood with AR DNA sequencing can help to improve the frequency of molecular diagnosis, particularly for partial AIS.

Interferon stimulated genes as peripheral diagnostic markers of early pregnancy in sheep: a critical assessment.

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0=day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 (CXCL10), Myxovirus (influenza virus) resistance 1 (MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher (P0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 (MX2) and ISG15 ubiquitin-like modifier (ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene® tubes on farms, for early pregnancy detection in sheep.

Real-time assessment of relapse risk based on the WT1 marker in acute leukemia and myelodysplastic syndrome patients after hematopoietic cell transplantation.

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT) for acute leukemia and myelodysplastic syndrome (MDS). Wilms' tumor Ag (WT1) is overexpressed in the majority of acute leukemia and MDS patients and has been proposed as a universal diagnostic marker for detection of impending relapse. Comprehensive studies have shown that WT1 transcript levels have predictive value in acute leukemia patients in CR after chemotherapy. However, the focus of this study is the period after alloHCT for predicting relapse onset. We analyzed the accumulation of WT1 mRNA transcripts in PB of 82 leukemia and MDS patients and defined specific molecular ratios for relapse prediction. The extensively validated WT1/c-ABL ratio was used to normalize increases in WT1 transcript levels. The observed lead time of crossing or exceeding set WT1 levels is presented along with linear interpolation to estimate the calculated day the WT1 thresholds were crossed. The WT1/c-ABL transcript ratio of 50 or above yielded 100% specificity and 75% sensitivity reliably predicting future relapse with an observed average of 29.4 days (s.d.=19.8) and a calculated average of 63 days (s.d.=29.3) lead time before morphologic confirmation. A lower ratio of 20 or above gave lower specificity, but higher sensitivity (84.8% and 87.5%, respectively) identified more patients who relapsed, at earlier times, providing an earlier warning with actual average lead time of 49.1 days (s.d.=30.8) and calculated average of 78 days (s.d.=28.8). WT1 transcript levels serve as a diagnostic relapse test with greater sensitivity than the morphologic approach used in the clinic as a readout.

Functional assessment of potential splice site variants in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

BACKGROUND: Interpretation of genetic screening results in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) often is difficult. Pathogenicity of variants with uncertain clinical significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants. OBJECTIVE: The purpose of this study was to perform functional analysis of potential splice site variants in ARVD/C patients. METHODS: Nine variants in desmosomal (PKP2, JUP, DSG2, DSC2) genes with potential RNA splicing effect were analyzed. The variants were found in patients who fulfilled 2010 ARVD/C Task Force Criteria (n = 7) or had suspected ARVD/C (n = 2). Total RNA was isolated from fresh blood samples and subjected to reverse transcriptase polymerase chain reaction. RESULTS: An effect on splicing was predicted by software algorithms for all variants. Of the 9 variants, 5 were intronic and 4 exonic. RNA analysis showed a functional effect on mRNA splicing by exon skipping, generation of new splice sites, or activation of cryptic sites in 6 variants. All 5 intronic variants tested severely impaired splicing. Only 1 of 4 exonic potential splice site variants was shown to have a deleterious effect on splicing. The remaining 3 exonic variants had no detectable effect on splicing, and heterozygous presence in mRNA confirmed biallelic expression. CONCLUSION: Six variants of uncertain clinical significance in the PKP2, JUP, and DSG2 genes showed a deleterious effect on mRNA splicing, indicating these are ARVD/C-related pathogenic splice site mutations. These results highlight the importance of functional assessment of potential splice site variants to improve patient care and facilitate cascade screening.

Posttransplantation bone marrow assessment by quantifying hematopoietic cell-derived mRNAs in plasma exosomes/microvesicles.

BACKGROUND: Bone marrow (BM) aspiration often can be a painful medical procedure. It is unavoidable, however, because hematopoietic precursor cells (HPC) exist only in BM and few escape to peripheral blood (PB). We hypothesized that HPCs might release exosomes and microvesicles (EMV) in BM, and the resulting EMV would penetrate into PB. Such BM-derived EMV might be identified in PB by measuring specific mRNAs produced by HPC. METHODS: Human plasma was applied to an EMV-capture filter plate. After centrifugation, captured EMV were lysed on the filter plate. Resulting lysates were transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by reverse transcription PCR (RT-PCR). Using this system, myeloid-, erythroid-, and megakaryocyte-lineage-specific poly(A)(+) mRNAs were quantified in plasma obtained from 18 patients who had undergone hematopoietic stem cell transplantation (HSCT). RESULTS: When fluorescent liposomes were applied to the filter plate, more than 95% of applied liposomes were absorbed. When human plasma was applied, a scanning electron microscope showed EMV-like particles on the membrane of the filter plate. After RT-PCR, various HPC-specific mRNAs were detected, and the results were equivalent to those derived from the standard ultracentrifugation method. The levels of these mRNAs were undetectable after HSCT and became detectable 1-2 weeks after HSCT, a substantially earlier time point than with traditional hematological analysis. The recovery of EMV mRNA at day 15 corresponded to the final clinical outcome at day 180. CONCLUSIONS: HPC-derived mRNAs in plasma EMV may represent new biomarkers for the assessment of BM condition and could reduce the necessity for frequent BM aspiration.

Development of blood biomarkers for drug-induced liver injury: an evaluation of their potential for risk assessment and diagnostics.

Drug-induced liver injury (DILI) remains a rare but serious complication in drug therapy that is a primary cause of drug failure during clinical trials. Conventional biomarkers, particularly the serum transaminases and bilirubin, serve as useful indicators of hepatocellular or cholestatic liver injury, respectively, but only after substantial and sometimes irreversible tissue damage. Ideally, more sensitive biomarkers that respond very early before irreversible injury has occurred would offer improved outcomes. Novel biomarkers are initially being developed in animal models exposed to intrinsically hepatotoxic stimuli. However, the eventual translation to human populations, even those with known risk factors that predispose the liver to drug toxicity, would be the fundamental goal. Ultimately, some might even be applicable for the early identification of individuals predisposed to idiosyncratic hepatotoxicity potential. This article reviews recent progress in the discovery and qualification of novel biomarkers for DILI and delineates the path to eventual utilization for risk assessment. Some major categories of plasma or serum biomarkers surveyed include proteins, cytokines, circulating mRNAs, and microRNAs.

Transforming growth factor ß1 protein and mRNA levels in inflammatory bowel diseases: towards solving the contradictions by longitudinal assessment of the protein and mRNA amounts.

Previously published studies on levels of the transforming growth factor-ß1 (TGF-ß1) protein and mRNA of the corresponding gene in patients suffering from inflammatory bowel diseases (IBD) gave varying results, leading to contradictory conclusions. To solve the contradictions, we aimed to assess longitudinally TGF-ß1 protein and mRNA levels at different stages of the disease in children suffering from IBD. The study group consisted of 19 pediatric patients with IBD at the age between 3.5 and 18.4 years. The control group consisted of 42 children aged between 2.0 and 18.0 years. The plasma TGF-ß1 concentration was measured with ELISA. mRNA levels of the TGF-ß1 gene isolated from samples of the intestinal tissue were assessed by reverse transcription and real-time PCR. Levels of TGF-ß1 protein in plasma and corresponding mRNA in intestinal tissue were significantly higher in IBD patients than in controls. TGF-ß1 and corresponding transcripts were also more abundant in plasma and intestinal tissue, respectively, in patients at the active stage of the disease than during remission. In every single IBD patient, plasma TGF-ß1 level and mRNA level in intestinal tissue was higher at the active stage of the disease than during remission. Levels of TGF-ß1 and corresponding mRNA are elevated during the active stage of IBD but not during the remission. Longitudinal assessment of this cytokine in a single patient may help to monitor the clinical course of IBD.

New insight into the pathogenesis of retinopathy of prematurity: assessment of whole-genome expression.

BACKGROUND: Retinopathy of prematurity (ROP) is one of the most common preventable causes of blindness and impaired vision among children in developed countries. The aim of the study was to compare whole-genome expression in the first month of life in groups of infants with and without ROP. METHODS: Blood samples were drawn from 111 newborns with a mean gestational age of 27.8 wk on the 5th, 14th, and 28th day of life (DOL). The mRNA samples were evaluated for gene expression with the use of human whole-genome microarrays. The infants were divided into two groups: no ROP (n = 61) and ROP (n = 50). RESULTS: Overall, 794 genes were differentially expressed on the 5th DOL, 1,077 on the 14th DOL, and 3,223 on the 28th DOL. In each of the three time points during the first month of life, more genes were underexpressed than overexpressed in the ROP group. Fold change (FC), which was used in analysis of gene expression data, ranged between 1.0 and 1.5 in the majority of genes differentially expressed. CONCLUSION: Pathway enrichment analysis revealed that genes in four pathways related to inflammatory response were consistently downregulated due to the following variables: ROP and gestational age.

Obesity, inflammation and resting energy expenditure: possible mechanism of progranulin in this pathway.

AIM: The aim of the study was to measure circulating PGRN levels and to investigate its potential correlation with resting metabolic rate and obesity related complications. Moreover, to investigate on the PGRN and some important gene expressions in energy expenditure in vitro in samples of PBMCs derived from all participants of our study in a cellular model. METHODS: Of the 163 participants who were recruited for the current cross-sectional study, 37 (22.69%) were normal weight (18.5≤BMI<25), 53 (32.51%) were overweight (25≤BMI<30), 48 (29.44%) were categorized as class I obese (BMI 30 -34.9) and 25 (15.33%) were classified as class II and III obese (BMI≥35). All participants were assessed for the measurement of RMR by means of indirect calorimetry following an overnight fasting. Body composition was analyzed with the Bioelectrical Impedance technique by the BODY COMPOSITION ANALYZER BC-418M -Tanita. The PBMCs were separated from whole blood by Ficoll-hypaque technique. Total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using specific primer pairs for PGRN, AKT, MAPK and mRNA, and beta actin mRNA was used as the internal control. Circulating PGRN was measured with the use of ELISA method. RESULTS: The circulating levels and gene expressions of PGRN rose in parallel with the increase of body weight. However, there was significant difference in the strength of association between circulating PGRN as well as PGRN gene expression and obesity-related variables. Moreover, PGRN gene expression had significant correlation with BMI, visceral fat, MAPK and AKT gene expression. The increased mass of visceral fat in correlation with the increased PGRN levels was more pronounced in high or normal resting metabolic rate group compared with the group with low resting metabolic rate. After adjusting for BMI and gender, we found that circulating PGRN can predict the RMR/kg independent of other variables such as TG, HDL, and hs-CRP (P=0.03). CONCLUSION: PGRN associated with obesity and glucose homeostasis and may predict the resting metabolic rate levels independent of confounder factors. Experimental study may clarify the PGRN role in obesity etiology through metabolism regulation.

Assessment of diabetic retinopathy by measuring retina-specific mRNA in blood.

INTRODUCTION: Diabetic retinopathy (DR) is the leading cause of blindness in adults. Early detection and treatment of DR has been shown to reduce the risk of visual loss by as much as 90%. At present, there are no blood tests to detect DR. Previous studies have demonstrated the presence of nucleic acids in blood and raised levels of these markers have been reported in many conditions. The aim of this study was to evaluate the circulating levels of retina-specific mRNA in the assessment of DR. AREAS COVERED: Blood samples were taken into PAXgene Blood RNA tubes from 89 diabetic patients and 19 healthy individuals. A reverse-transcription quantitative real-time PCR assay was used to measure circulating levels of mRNA for rhodopsin (Rho), retinal amine oxidase (RAO) and phosphodiesterase 6C (PDE6C). The results were normalized against mRNA for ß-actin and total RNA. While mRNA for Rho, RAO and ß-actin were detected in 100% of the subjects, PDE6C mRNA was only found in 60% of the individuals and melanopsin mRNA was not detected. When diabetic subjects were divided according to their DR status, significant differences were observed for Rho and RAO-Rho increased while RAO tended to decrease. The area under the curve ROC for Rho and Rho/RAO ratio to differentiate mild or no DR from significant DR (pre-proliferative and proliferative stages) were 0.756 and 0.823, respectively. EXPERT OPINION: These findings suggest that circulating mRNA may be useful in assessing DR.

Assessment of the effect of surgery on the kinetics of circulating tumour cells in patients with operable breast cancer based on cytokeratin-19 mRNA detection.

AIMS: The aim of this study was to evaluate the effect of surgery on the kinetics of CTCs in breast cancer patients. METHODS: The detection of CK-19 mRNA-positive CTCs in the blood by RT-PCR was analysed in 104 stage 0-IIIA patients at 4 time-points: prior to surgery, upon completion, 24 h after surgery and 15 days after surgery. Furthermore, a late sample was assessed prior to initiation of adjuvant chemotherapy in a subgroup of 53 patients. As negative controls, peripheral blood was obtained from 50 female patients undergoing excision of benign breast lesions and from 11 female patients receiving surgery for early-stage colorectal cancer. RESULTS: A significant percentage of blood samples from breast cancer patients (14.4%) were negative for CK-19 preoperatively but turned transiently positive early postoperatively. However, no significant difference in CK-19 mRNA detection was noted among the first 4 examined time-points. There was no significant correlation between CK-19 mRNA-positive cells and classic prognostic factors. A significant increase in CK-19 mRNA-positivity (32.1%) was observed in a late sample of the subgroup of 53 patients before adjuvant chemotherapy after a median of 54 days, postoperatively. CONCLUSIONS: Surgery may result in CTC detection in a small proportion of early breast cancer patients. There is no clear correlation to indicate which patients are expected to have detectable CTCs. Although CTCs are detected in a small proportion of patients during the perioperative period, the detection rate may increase over time and with longer follow-up.

Preclinical safety assessment: current gaps, challenges, and approaches in identifying translatable biomarkers of drug-induced liver injury.

Currently, no serum biomarkers, including the biochemical gold standard alanine aminotransferase, can differentiate drug-induced from non-drug-related liver injury, can differentiate liver injury mediated by a specific drug or mechanism, or can accurately predict the progression and outcome of hepatic injury. Efforts have been made by veterinary clinical pathologists, toxicologists, and other scientists to address the gaps in hepatic biomarkers faced during drug development; although there have been no breakthroughs, several novel biomarker candidates have been identified. Efforts to address the gaps in translatable hepatic biomarkers and the challenges and hurdles faced during this process are highlighted in this review.