Deep Learning-Based Knee MRI Classification for Common Peroneal Nerve Palsy with Foot Drop.

Foot drop can have a variety of causes, including the common peroneal nerve (CPN) injuries, and is often difficult to diagnose. We aimed to develop a deep learning-based algorithm that can classify foot drop with CPN injury in patients with knee MRI axial images only. In this retrospective study, we included 945 MR image data from foot drop patients confirmed with CPN injury in electrophysiologic tests (n = 42), and 1341 MR image data with non-traumatic knee pain (n = 107). Data were split into training, validation, and test datasets using a 8:1:1 ratio. We used a convolution neural network-based algorithm (EfficientNet-B5, ResNet152, VGG19) for the classification between the CPN injury group and the others. Performance of each classification algorithm used the area under the receiver operating characteristic curve (AUC). In classifying CPN MR images and non-CPN MR images, EfficientNet-B5 had the highest performance (AUC = 0.946), followed by the ResNet152 and the VGG19 algorithms. On comparison of other performance metrics including precision, recall, accuracy, and F1 score, EfficientNet-B5 had the best performance of the three algorithms. In a saliency map, the EfficientNet-B5 algorithm focused on the nerve area to detect CPN injury. In conclusion, deep learning-based analysis of knee MR images can successfully differentiate CPN injury from other etiologies in patients with foot drop.

Predictive Modeling and Integrated Risk Assessment of Postoperative Mortality and Pneumonia in Traumatic Brain Injury Patients through Clustering and Machine Learning: Retrospective Study.

This study harnessed machine learning to forecast postoperative mortality (POM) and postoperative pneumonia (PPN) among surgical traumatic brain injury (TBI) patients. Our analysis centered on the following key variables: Glasgow Coma Scale (GCS), midline brain shift (MSB), and time from injury to emergency room arrival (TIE). Additionally, we introduced innovative clustered variables to enhance predictive accuracy and risk assessment. Exploring data from 617 patients spanning 2012 to 2022, we observed that 22.9% encountered postoperative mortality, while 30.0% faced postoperative pneumonia (PPN). Sensitivity for POM and PPN prediction, before incorporating clustering, was in the ranges of 0.43-0.82 (POM) and 0.54-0.76 (PPN). Following clustering, sensitivity values were 0.47-0.76 (POM) and 0.61-0.77 (PPN). Accuracy was in the ranges of 0.67-0.76 (POM) and 0.70-0.81 (PPN) prior to clustering and 0.42-0.73 (POM) and 0.55-0.73 (PPN) after clustering. Clusters characterized by low GCS, small MSB, and short TIE exhibited a 3.2-fold higher POM risk compared to clusters with high GCS, small MSB, and short TIE. In summary, leveraging clustered variables offers a novel avenue for predicting POM and PPN in TBI patients. Assessing the amalgamated impact of GCS, MSB, and TIE characteristics provides valuable insights for clinical decision making.

CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

Gene expression is changed by disease, but how these molecular responses arise and contribute to pathophysiology remains less understood. We discover that ß-amyloid, a trigger of Alzheimer's disease (AD), promotes the formation of pathological CREB3L2-ATF4 transcription factor heterodimers in neurons. Through a multilevel approach based on AD datasets and a novel chemogenetic method that resolves the genomic binding profile of dimeric transcription factors (ChIPmera), we find that CREB3L2-ATF4 activates a transcription network that interacts with roughly half of the genes differentially expressed in AD, including subsets associated with ß-amyloid and tau neuropathologies. CREB3L2-ATF4 activation drives tau hyperphosphorylation and secretion in neurons, in addition to misregulating the retromer, an endosomal complex linked to AD pathogenesis. We further provide evidence for increased heterodimer signaling in AD brain and identify dovitinib as a candidate molecule for normalizing ß-amyloid-mediated transcriptional responses. The findings overall reveal differential transcription factor dimerization as a mechanism linking disease stimuli to the development of pathogenic cellular states.

A systemic cell stress signal confers neuronal resilience toward oxidative stress in a Hedgehog-dependent manner.

Cells possess several conserved adaptive mechanisms to respond to stress. Stress signaling is initiated to reestablish cellular homeostasis, but its effects on the tissue or systemic levels are far less understood. We report that the secreted luminal domain of the endoplasmic reticulum (ER) stress transducer CREB3L2 (which we name TAILS [transmissible activator of increased cell livability under stress]) is an endogenous, cell non-autonomous activator of neuronal resilience. In response to oxidative insults, neurons secrete TAILS, which potentiates hedgehog signaling through direct interaction with Sonic hedgehog (SHH) and its receptor PTCH1, leading to improved antioxidant signaling and mitochondrial function in neighboring neurons. In an in vivo model of ischemic brain injury, administration of TAILS enables survival of CNS neurons and fully preserves cognitive function in behavioral tests. Our findings reveal an SHH-mediated, cell non-autonomous branch of cellular stress signaling that confers resilience to oxidative stress in the mature brain, providing protection from ischemic neurodegeneration.

New Genotypes and Diversity of Orientia tsutsugamushi DNA samples from Patients with Scrub Typhus in South Korea as Determined by Multilocus Sequence Typing.

Orientia tsutsugamushi, an obligate intracellular organism, is the causative agent of scrub typhus. Multilocus sequence typing (MLST) is a genetic typing method that provides a unified bacterial isolate characterization approach. However, there are no comparative studies in South Korea on the genotypic properties of O. tsutsugamushi based on MLST. To conduct a comparative analysis with previous data collected from Thailand, Laos, and Japan, we investigated the genetic diversity of O. tsutsugamushi from 51 patients with scrub typhus in South Korea by using MLST. The MLST analysis revealed 10 new alleles in the housekeeping genes: gpsA, n = 2; mdh, n = 1; nrdB, n = 1; nuoF, n = 1; ppdK, n = 1; sucB, n = 2; and sucD, n = 2. These novel alleles led to the assignment of six new sequence types (STs) (ST93-98). The 51 samples corresponded to seven different STs (ST48 and ST93-98), with ST48 accounting for the largest proportion (49.0%) of O. tsutsugamushi STs in South Korea. Interestingly, O. tsutsugamushi from patients with scrub typhus in South Korea were clustered in two different clades, and the five Korean STs (ST48, ST93, ST94, ST95, and ST98) were close genetically to ST80, which was isolated from Laos. The remaining two STs (ST96 and ST97) were close genetically to ST49 (Ikeda, Japan). Overall, our results suggest that the relative genetic stability and the clonal populations of O. tsutsugamushi strains in South Korea have remained mostly conserved.

Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERBα.

Hyperimmunity drives the development of Alzheimer disease (AD). The immune system is under the circadian control, and circadian abnormalities aggravate AD progress. Here, we investigate how an AD-linked mutation deregulates expression of circadian genes and induces cognitive decline using the knock-in (KI) mice heterozygous for presenilin 2 N141I mutation. This mutation causes selective overproduction of clock gene-controlled cytokines through the DNA hypermethylation-mediated repression of REV-ERBα in innate immune cells. The KI/+ mice are vulnerable to otherwise innocuous, mild immune challenges. The antipsychotic chlorpromazine restores the REV-ERBα level by normalizing DNA methylation through the inhibition of PI3K/AKT1 pathway, and prevents the overexcitation of innate immune cells and cognitive decline in KI/+ mice. These results highlight a pathogenic link between this AD mutation and immune cell overactivation through the epigenetic suppression of REV-ERBα.

GSK3B induces autophagy by phosphorylating ULK1.

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis.

A MARTX Toxin rtxA Gene Is Controlled by Host Environmental Signals through a CRP-Coordinated Regulatory Network in Vibrio vulnificus.

A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, P rtxA Phased hypersensitivity resulting from DNase I cleavage of the P rtxA regulatory region suggests that Lrp probably induces DNA bending in P rtxA Lrp activates P rtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to P rtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses P rtxA , and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of P rtxA , which presumably alters the DNA conformation in P rtxA and thus represses rtxA Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection.IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail.

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

Novel short peptide tag from a bacterial toxin for versatile applications.

The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTXVv. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection in enzyme-linked immunosorbent, Western blot, flow cytometry, and immunofluorescence staining analyses in Escherichia coli, mammalian cell lines, yeast, and plant. In addition, our RA-tag/47RA mAb combination showed high sensitivity and reliable affinity (KD = 5.90 × 10-8 M) when compared with conventional tags. Overall, our results suggest that the RA-tag system could facilitate the development of a broadly applicable tag system for biological research.

CASP9 (caspase 9) is essential for autophagosome maturation through regulation of mitochondrial homeostasis.

CASP9 (caspase 9) is a well-known initiator caspase which triggers intrinsic apoptosis. Recent studies also suggest various non-apoptotic roles of CASP9, including macroautophagy/autophagy regulation. However, the involvement of CASP9 in autophagy and its molecular mechanisms are not well understood. Here we report the non-apoptotic function of CASP9 in positive regulation of autophagy through maintenance of mitochondrial homeostasis. Growth factor or amino acid deprivation-induced autophagy activated CASP9, but without apoptotic features. Pharmacological inhibition or genetic ablation of CASP9 decreased autophagy flux, while ectopic expression of CASP9 rescued autophagy defects. In CASP9 knockout (KO) cells, initiation and elongation of phagophore membranes were normal, but sealing of the membranes and autophagosome maturation were impaired, and the lifetime of autophagosomes was prolonged. Ablation of CASP9 caused an accumulation of inactive ATG3 and decreased lipidation of the Atg8-family members, most severely that of GABARAPL1. Moreover, it resulted in abnormal mitochondrial morphology with depolarization of the membrane potential, reduced reactive oxygen species production, and aberrant accumulation of mitochondrial fusion-fission proteins. CASP9 expression or exogenously added H2O2 in the CASP9 KO cells corrected the ATG3 level and lipidation status of Atg8-family members, and restored autophagy flux. Of note, only CASP9 expression but not H2O2 rescued mitochondrial defects, revealing regulation of mitochondrial homeostasis by CASP9. Our findings suggest a new regulatory link between mitochondria and autophagy through CASP9 activity, especially for the proper operation of the Atg8-family conjugation system and autophagosome closure and maturation. ABBREVIATIONS: AA: amino acid; ACD: autophagic cell death; ACTB: actin beta; ANXA5: annexin A5; APAF1: apoptotic peptidase activating factor 1; Atg: autophagy related; ATG16L1: autophagy related 16 like 1; BafA1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; CARD: caspase recruitment domain containing; CASP: caspase; CM-H2DCFDA: chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; Δψm: mitochondrial membrane potential; DN: dominant-negative; DNM1L/DRP1: dynamin 1 like; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; HCN: hippocampal neural stem cells; IAM: inner autophagosome membrane; INS: insulin; KO: knockout; LEHD: Z-LEHD-fmk; MAP1LC3: microtubule associated protein 1 light chain 3; MFN1: mitofusin 1; MFN2: mitofusin 2; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP1: poly(ADP-ribose) polymerase 1; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; ROS: reactive oxygen species; sgRNA: single guide RNA; SR-SIM: super-resolution structured illumination microscopy; SQSTM1: sequestosome 1; STS: staurosporine; STX17: syntaxin 17; TMRE: tetramethylrhodamine ethyl ester; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Promotion of Axon Growth by the Secreted End of a Transcription Factor.

Axon growth is regulated externally by attractive and repulsive cues generated in the environment. In addition, intrinsic pathways govern axon development, although the extent to which axons themselves can influence their own growth is unknown. We find that dorsal root ganglion (DRG) axons secrete a factor supporting axon growth and identify it as the C terminus of the ER stress-induced transcription factor CREB3L2, which is generated by site 2 protease (S2P) cleavage in sensory neurons. S2P and CREB3L2 knockdown or inhibition of axonal S2P interfere with the growth of axons, and C-terminal CREB3L2 is sufficient to rescue these effects. C-terminal CREB3L2 forms a complex with Shh and stabilizes its association with the Patched-1 receptor on developing axons. Our results reveal a neuron-intrinsic pathway downstream of S2P that promotes axon growth.

Parkin Promotes Mitophagic Cell Death in Adult Hippocampal Neural Stem Cells Following Insulin Withdrawal.

Regulated cell death (RCD) plays a fundamental role in human health and disease. Apoptosis is the best-studied mode of RCD, but the importance of other modes has recently been gaining attention. We have previously demonstrated that adult rat hippocampal neural stem (HCN) cells undergo autophagy-dependent cell death (ADCD) following insulin withdrawal. Here, we show that Parkin mediates mitophagy and ADCD in insulin-deprived HCN cells. Insulin withdrawal increased the amount of depolarized mitochondria and their colocalization with autophagosomes. Insulin withdrawal also upregulated both mRNA and protein levels of Parkin, gene knockout of which prevented mitophagy and ADCD. c-Jun is a transcriptional repressor of Parkin and is degraded by the proteasome following insulin withdrawal. In insulin-deprived HCN cells, Parkin is required for Ca2+ accumulation and depolarization of mitochondria at the early stages of mitophagy as well as for recognition and removal of depolarized mitochondria at later stages. In contrast to the pro-death role of Parkin during mitophagy, Parkin deletion rendered HCN cells susceptible to apoptosis, revealing distinct roles of Parkin depending on different modes of RCD. Taken together, these results indicate that Parkin is required for the induction of ADCD accompanying mitochondrial dysfunction in HCN cells following insulin withdrawal. Since impaired insulin signaling is implicated in hippocampal deficits in various neurodegenerative diseases and psychological disorders, these findings may help to understand the mechanisms underlying death of neural stem cells and develop novel therapeutic strategies aiming to improve neurogenesis and survival of neural stem cells.

Alzheimer's disease and the autophagic-lysosomal system.

Age-related neurodegenerative diseases are of critical concern to the general population and research/medical community due to their health impact and socioeconomic consequences. A feature of most, if not all, neurodegenerative disorders is the presence of proteinopathies, in which misfolded or conformationally altered proteins drive disease progression and are often used as a primary neuropathological marker of disease. In particular, Alzheimer's disease (AD) is characterized by abnormal accumulation of protein aggregates, primarily extracellular plaques composed of the Aß peptide and intracellular tangles comprised of the tau protein, both of which may indicate a primary defect in protein clearance. Protein degradation is a key cellular mechanism for protein homeostasis and is essential for cell survival but is disrupted in neurodegenerative diseases. Dysregulation in proteolytic pathways - mainly the autophagic-lysosomal system (A-LS) and the ubiquitin-proteasome system (UPS) - has been increasingly associated with proteinopathies in neurodegenerative diseases. Here we review the role of dysfunctional autophagy underlying AD-related proteinopathy and discuss how to model this aspect of disease, as well as summarize recent advances in translational strategies for targeted A-LS dysfunction in AD.

ArmA and RmtB Were the Predominant 16S RMTase Genes Responsible for Aminoglycoside-resistant Isolates in Korea.

Pathogenic gram-negatives that produce 16S ribosomal RNA methyltransferases (16S RMTases) have already been distributed all over the world. To investigate the predominance of aminoglycoside resistance associated with 16S RMTases in Korea, we collected a total of 222 amikacin resistant Gram-negative clinical isolates from patient specimens between 1999 and 2015 from three hospital banks across Korea. ArmA and rmtB were the predominant 16S RMTase genes responsible for aminoglycoside-resistant isolates circulating in Korean community settings although only one rmtA-producing isolate was detected in 2006.

The NETest: The Clinical Utility of Multigene Blood Analysis in the Diagnosis and Management of Neuroendocrine Tumors.

The neuroendocrine neoplasms test (NETest) is a multianalyte liquid biopsy that measures neuroendocrine tumor gene expression in blood. This unique signature precisely defines the biological activity of an individual tumor in real time. The assay meets the 3 critical requirements of an optimal biomarker: diagnostic accuracy, prognostic value, and predictive therapeutic assessment. NETest performance metrics are sensitivity and specificity and in head-to-head comparison are 4-fold to 10-fold more accurate than chromogranin A. NETest accurately identifies completeness of surgery and response to somatostatin analogs. Clinical registry data demonstrate significant clinical utility in watch/wait programs.

A liquid biopsy for bronchopulmonary/lung carcinoid diagnosis.

No effective blood biomarker exists to detect and clinically manage bronchopulmonary (BP) neuroendocrine tumors (NET). We developed a blood-based 51 NET-specific transcript set for diagnosis and monitoring and evaluated clinical performance metrics. It accurately diagnosed the tumor and differentiated stable from progressive disease as determined by RECIST criteria. Gene expression was evaluated in: a) publicly available BPNET transcriptomes (GSE35679); b) two BPNET cell-lines; and c) BPNET tissue with paired blood (n = 7). Blood gene expression was assessed in 194 samples including controls, benign lung diseases, malignant lung diseases and small bowel NETs. A separate validation study in 25 age- and gender-matched BPNETs/controls was performed. Gene expression measured by real-time PCR was scored (0-100%; normal: < 14%). Regression analyses, Principal Component Analysis (PCA), hierarchical clustering, Fisher's and non-parametric evaluations were undertaken. All 51 genes were identified in BPNET transcriptomes, tumor samples and cell-lines. Significant correlations were evident between paired tumor and blood (R2:0.63-0.91, p < 0.001). PCA and hierarchical clustering identified blood gene expression was significantly different between lung cancers and benign diseases, including BPNETs. Gene expression was highly correlated (R2: 0.91, p = 1.7 × 10-15) between small bowel and BPNET. For validation, all 25 BPNETs were positive compared to 20% controls (p < 0.0001). Scores were significantly elevated (p < 0.0001) in BPNETs (57 ± 28%) compared to controls (4 ± 5%). BPNETs with progressive disease (85 ± 11%) exhibited higher scores than stable disease (32 ± 7%, p < 0.0001). Blood measurements accurately diagnosed bronchopulmonary carcinoids, distinguishing stable from progressive disease. This marker panel will have clinical utility as a diagnostic liquid biopsy able to define disease activity and progression in real-time.

In vitro and in vivo Inhibitory Activity of NADPH Against the AmpC BER Class C ß-Lactamase.

ß-Lactamase-mediated resistance to ß-lactam antibiotics has been significantly threatening the efficacy of these clinically important antibacterial drugs. Although some ß-lactamase inhibitors are prescribed in combination with ß-lactam antibiotics to overcome this resistance, the emergence of enzymes resistant to current inhibitors necessitates the development of novel ß-lactamase inhibitors. In this study, we evaluated the inhibitory effect of dinucleotides on an extended-spectrum class C ß-lactamase, AmpC BER. Of the dinucleotides tested, NADPH, a cellular metabolite, decreased the nitrocefin-hydrolyzing activity of the enzyme with a Ki value of 103 µM in a non-covalent competitive manner. In addition, the dissociation constant (KD) between AmpC BER and NADPH was measured to be 40 µM. According to our in vitro susceptibility study based on growth curves, NADPH restored the antibacterial activity of ceftazidime against a ceftazidime-resistant Escherichia coli BER strain producing AmpC BER. Remarkably, a single dose of combinatory treatment with NADPH and ceftazidime conferred marked therapeutic efficacy (100% survival rate) in a mouse model infected by the E. coli BER strain although NADPH or ceftazidime alone failed to prevent the lethal bacterial infection. These results may offer the potential of the dinucleotide scaffold for the development of novel ß-lactamase inhibitors.

The utility of blood neuroendocrine gene transcript measurement in the diagnosis of bronchopulmonary neuroendocrine tumours and as a tool to evaluate surgical resection and disease progression.

OBJECTIVES: The management of bronchopulmonary neuroendocrine tumours (BPNETs) is difficult, since imaging, histology and biomarkers have a limited value in diagnosis, predicting outcome and defining therapeutic efficacy. We evaluated a NET multigene blood test (NETest) to diagnose BPNETs, assess disease status and evaluate surgical resection. METHODS: (i) Diagnostic cohort: BP carcinoids (n = 118)-typical carcinoid, n = 67 and atypical carcinoid, n = 51; other lung NEN (large-cell neuroendocrine carcinoma and small-cell lung carcinoma, n = 13); adenocarcinoma, (n = 26); squamous cell carcinoma (n = 23); controls (n = 90) and chronic obstructive pulmonary disease (n = 18). (ii) Surgical cohort, n = 28: BP carcinoids (n = 16: typical carcinoid 12; atypical carcinoid 4); large-cell neuroendocrine carcinoma, n = 3; lung adenocarcinoma, n = 8 and squamous cell carcinoma, n = 1. Blood sampling was performed presurgery and 30 days post-surgery. Transcript levels measured by quantitative polymerase chain reaction were calculated as activity scores (0-100% scale: normal < 14%) and compared with chromogranin A (enzyme-linked immunosorbent assay; normal <109 ng/ml). RESULTS: NETest was significantly elevated in carcinoids (48.7 ± 27%) versus controls (6 ± 6%, P < 0.001) with metrics: sensitivity 93%, specificity 89%, positive predictive value 92% and negative predictive value 91%. NETest differentiated progressive disease (73 ± 22%) from stable disease (36 ± 19%, P < 0.001) and R0 resections (10 ± 5%, P < 0.001, area under the curve: 0.98). Levels in chronic obstructive pulmonary disease and lung cancers were 18-24% while elevated in small-cell lung carcinoma/large-cell neuroendocrine carcinoma (59 ± 10%). In BPNETs on postoperative Day 30, NETest decreased by 60% (P < 0.001). Chromogranin A was elevated in only 40% of carcinoids and not altered by surgery. CONCLUSIONS: Blood NET gene levels accurately identified BPNETs (100%) and differentiated these from controls, benign and malignant lung disease. Progressive disease could be identified and surgical resection verified. Chromogranin A had no clinical utility. Monitoring NET transcript levels in blood will facilitate management by detecting residual tumour and identifying progressive disease.

Molecular strategies in the management of bronchopulmonary and thymic neuroendocrine neoplasms.

Thoracic NETs [bronchopulmonary NETs (BPNETs) and thymic NETs (TNET)] share a common anatomic primary location, likely a common cell of origin, the "Kulchitsky cell" and presumably, a common etiopathogenesis. Although they are similarly grouped into well-differentiated [typical carcinoids (TC) and atypical carcinoids (AC)] and poorly differentiated neoplasms and both express somatostatin receptors, they exhibit a wide variation in clinical behavior. TNETs are more aggressive, are frequently metastatic, and have a lower 5-year survival rate (~50% vs. ~80%) than BPNETs. They are typically symptomatic, most often secreting ACTH (40% of tumors) but both tumor groups share secretion of common biomarkers including chromogranin A and 5-HIAA. Consistently effective and accurate circulating biomarkers are, however, currently unavailable. Surgery is the primary therapeutic tool for both BPNET and TNETs but there remains little consensus about later interventions e.g., targeted therapy, or how these can be monitored. Genetic analyses have identified different topographies (e.g., significant alterations in chromatin and epigenetic remodeling in BPNETs versus frequent chromosomal abnormalities in TNETs) but there is an absence of clinically actionable mutations in both tumor groups. Liquid biopsies, tools that can measure neoplastic signatures in peripheral blood, can potentially be leveraged to detect disease early i.e., recurrence, predict tumors that may respond to specific therapies and serve as real-time monitors for treatment responses. Recent studies have identified that mRNA transcript analysis in blood effectively identifies both BPNET and TNETs. The clinical utility of this gene expression assay includes use as a diagnostic, confirmation of completeness of surgical resection and use as a molecular management tool to monitor efficacy of PRRT and other therapeutic strategies.