Pneumonic-type lung adenocarcinoma with KRAS G12V mutation and sustained response to Afatinib.

BACKGROUND: Pneumonic-type lung adenocarcinoma (P-ADC) is a rare and challenging subtype of primary lung cancer that can be difficult to distinguish from pneumonia based on radiological images. Furthermore, no drugs are currently available that specifically target KRAS G12V. CASE PRESENTATION: Here we report a case of P-ADC with typical and informative imaging features throughout the course of the disease, including patchy shadows, high-density lesions with aerated bronchus, diffuse ground-glass opacities, and nodular shadows from computed tomography (CT) scan. The KRAS G12V mutation was detected using Next-generation sequencing (NGS). An individualized Afatinib-based therapeutic schedule was prescribed and achieved sustained response after multiple lines of treatment had failed. CONCLUSION: Our case highlights the typical and dynamic changes in imaging features of P-ADC and provides an indicative treatment strategy for KRAS G12V-mutated lung adenocarcinoma.

An Unusual Presentation of Cobblestone Esophagus From Bisphosphonate Use.

Cobblestone esophagus is a rare finding that has been previously described in cases of eosinophilic esophagitis (EoE), candidiasis, Barrett's esophagus, or severe reflux esophagitis from distal gastrointestinal obstruction. We describe a case of asymptomatic cobblestone esophagus secondary to bisphosphonate use.  A 67-year-old female was seen in the clinic for evaluation of microcytic anemia that was incidentally picked up on routine chronic disease follow-up. She had no gastrointestinal symptoms. She has been taking oral alendronate 70mg once a week for osteoporosis since a year ago. Barium meal was performed as the patient initially opted for non-invasive testing, which incidentally showed a diffuse "cobblestone" appearance. Subsequent oesophago-gastro-duodenoscopy (OGD) showed diffuse white nodular lesions along the esophagus with a cobblestone appearance but no ulcer or mass. Segmental esophageal biopsies were negative for fungal stain and did not show any pathology. In the absence of infection, eosinophilic esophagitis, and dysplasia, her "cobblestone" esophagus was attributed to bisphosphonate use by diagnosis of exclusion. Alendronate acid was held off, and serial barium meals over the next year showed significant interval improvement.  Bisphosphonates, such as alendronate acid, are commonly associated with drug-induced esophagitis. With the cessation of the offending medication, there was indeed a significant improvement in our patient's serial barium meal. It is important to review the medication list when encountering patients who present with cobblestone esophagus, as some of these patients with drug-induced esophagitis may be asymptomatic clinically.

MET alterations detection platforms and clinical implications in solid tumors: a comprehensive review of literature.

MET alterations, including MET exon 14 skipping variants, MET amplification, MET overexpression, and MET fusion, play pivotal roles in primary tumorigenesis and acquired resistance to targeted therapies, especially EGFR tyrosine kinase inhibitors. They represent important diagnostic, prognostic, and predictive biomarkers in many solid tumor types. However, the detection of MET alterations is challenging due to the complexity of MET alterations and the diversity of platform technologies. Therefore, techniques with high sensitivity, specificity, and reliable molecular detection accuracy are needed to overcome such hindrances and aid in biomarker-guided therapies. The current review emphasizes the role of MET alterations as oncogenic drivers in a variety of cancers and their involvement in the development of resistance to targeted therapies. Moreover, our review provides an overview of and recommendations on the selection of various cross-platform technologies for the detection of MET exon 14 skipping variants, MET amplification, MET overexpression, and MET fusion. Furthermore, challenges and hurdles underlying these common detection platforms are discussed.

Importance of N-Glycosylation for the Expression and Function of Human Organic Anion Transporting Polypeptide 2B1.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a membrane transporter widely expressed in organs crucial for drug absorption and disposition such as the intestine, liver, and kidney. Evidence indicates that OATP2B1 is a glycoprotein. However, the sites of glycosylation and their contribution to the function and expression of OATP2B1 are largely unknown. In this study, by site-directed mutagenesis, we determined that two of four potential N-glycosylation sites in OATP2B1, N176 and N538, are indeed glycosylated. Functional studies revealed that the transport activities of mutants N176Q and N538Q were greatly reduced as compared to that of wild-type OATP2B1. However, the reduced activity was not due to the impairment of transport function per se but due to the decreased surface expression as the Km and normalized Vmax values of N176Q and N538Q were comparable to those of OATP2B1. Quantitative polymerase chain reaction (PCR) revealed that N176Q and N538Q mutations did not affect the expression of OATP2B1 at a transcriptional level. Immunofluorescence analysis showed that deglycosylated OATP2B1 was largely retained in the endoplasmic reticulum, which may activate the endoplasmic reticulum-associated degradation pathway, and the ubiquitin-proteasome system played a major role in the degradation of OATP2B1. Taken together, OATP2B1 is N-glycosylated, and N-glycosylation is essential for the surface expression of OATP2B1 but not critical for the transport function of OATP2B1 per se.

Regulation of ERK2 activity by dynamic S-acylation.

Extracellular signal-regulated kinases (ERK1/2) are key effector proteins of the mitogen-activated protein kinase pathway, choreographing essential processes of cellular physiology. Here, we discover that ERK1/2 are subject to S-acylation, a reversible lipid modification of cysteine residues, at C271/C254. The levels of ERK1/2 S-acylation are modulated by epidermal growth factor (EGF) signaling, mirroring its phosphorylation dynamics, and acylation-deficient ERK2 displays altered phosphorylation patterns. We show that ERK1/2 S-acylation is mediated by "writer" protein acyl transferases (PATs) and "eraser" acyl protein thioesterases (APTs) and that chemical inhibition of either lipid addition or removal alters ERK1/2's EGF-triggered transcriptional program. Finally, in a mouse model of metabolic syndrome, we find that ERK1/2 lipidation levels correlate with alterations in ERK1/2 lipidation writer/eraser expression, solidifying a link between ERK1/2 activity, ERK1/2 lipidation, and organismal health. This study describes how lipidation regulates ERK1/2 and offers insight into the role of dynamic S-acylation in cell signaling more broadly.

FDW028, a novel FUT8 inhibitor, impels lysosomal proteolysis of B7-H3 via chaperone-mediated autophagy pathway and exhibits potent efficacy against metastatic colorectal cancer.

Metastatic colorectal cancer (mCRC) is a major cause of cancer-related mortality due to the absence of effective therapeutics. Thus, it is urgent to discover new drugs for mCRC. Fucosyltransferase 8 (FUT8) is a potential therapeutic target with high level in most malignant cancers including CRC. FUT8 mediates the core fucosylation of CD276 (B7-H3), a key immune checkpoint molecule (ICM), in CRC. FUT8-silence-induced defucosylation at N104 on B7-H3 attracts heat shock protein family A member 8 (HSPA8, also known as HSC70) to bind with 106-110 SLRLQ motif and consequently propels lysosomal proteolysis of B7-H3 through the chaperone-mediated autophagy (CMA) pathway. Then we report the development and characterization of a potent and highly selective small-molecule inhibitor of FUT8, named FDW028, which evidently prolongs the survival of mice with CRC pulmonary metastases (CRPM). FDW028 exhibits potent anti-tumor activity by defucosylation and impelling lysosomal degradation of B7-H3 through the CMA pathway. Taken together, FUT8 inhibition destabilizes B7-H3 through CMA-mediated lysosomal proteolysis, and FDW028 acts as a potent therapeutic candidate against mCRC by targeting FUT8. FDW028, an inhibitor specifically targeted FUT8, promotes defucosylation and consequent HSC70/LAMP2A-mediated lysosomal degradation of B7-H3, and exhibits potent anti-mCRC activities.

Clinicopathological features of incidentally detected metastatic thyroid papillary carcinoma in cervical lymph nodes of non-thyroid cancer patients: a retrospective analysis of 31cases.

BACKGROUND: The incidental finding of thyroid inclusions in lymph nodes of neck dissections of non-thyroid cancer patients is an unusual event. It is still controversial for pathologists about whether this represents benign inclusions or metastatic papillary thyroid carcinoma (PTC). This study is to analyze clinicopathological features of such cases in an attempt to explore their clinical implications. METHODS: Pathological data were searched for incidentally detected PTC of cervical lymph nodes in non-thyroid cancer cases. Clinicopathological characteristics were reevaluated and recorded. BRAF V600E protein expression and sequencing analysis was then performed in cases with sufficient tissues. RESULTS: 31 patients had an incidental finding of PTC in lymph nodes of patients with non-thyroid cancer. BRAF immunohistochemical staining were performed in 17 metastatic lymph nodes with sufficient tumor tissues, and 6 were positive. BRAF V600E point mutation was detected in 5 of 6 BRAF V600E positive cases. Subsequent imaging examinations of the thyroid showed no nodules or calcifications/benign nodules in 20 patients, and suspected malignant nodules in 5 patients. 12 patients underwent total thyroidectomy or ipsilateral lobectomy, and 6 showed PTC in postoperative pathological examinations. The remaining 19 patients without surgery were kept under active surveillance, and no one had recurrence of PTC. CONCLUSION: Incidentally discovered PTC in lymph nodes has usually interpreted as metastasis from a clinical occult thyroid primary cancer, but primary PTC was not always detected. This suggests it could be double occult lesions. With regards to concurrence with highly malignant tumor, most patients could keep regular surveillance.

Corrosion Behavior of Alumina-Forming Austenitic Steel in Supercritical Carbon Dioxide Conditions: Effects of Nb Content and Temperature.

The corrosion behavior of alumina-forming austenitic (AFA) stainless steels with different Nb additions in a supercritical carbon dioxide environment at 500 °C, 600 °C, and 20 MPa was investigated. The steels with low Nb content were found to have a novel structure with a double oxide as an outer Cr2O3 oxide film and an inner Al2O3 oxide layer with discontinuous Fe-rich spinels on the outer surface and a transition layer consisting of Cr spinels and γ'-Ni3Al phases randomly distributed under the oxide layer. Oxidation resistance was improved by accelerating diffusion through refined grain boundaries after the addition of 0.6 wt.% Nb. However, the corrosion resistance decreased significantly at higher Nb content due to the formation of continuous thick outer Fe-rich nodules on the surface and an internal oxide zone, and Fe2(Mo, Nb) laves phases were also detected, which prevented the outward diffusion of Al ions and promoted the formation of cracks within the oxide layer, resulting in unfavorable effects on oxidation. After exposure at 500 °C, fewer spinels and thinner oxide scales were found. The specific mechanism was discussed.

Ti3C2 and Ti2C MXene materials for high-performance isolation of extracellular vesicles via coprecipitation.

Two-dimensional (2D) materials such as MXenes, are usually well utilized in the field of catalysts and battery due to their good hydrophilicity and diversified surface terminals. However, their potential applications in the treatment of biological samples have not been widely concerned. Extracellular vesicles (EVs) contain unique molecular signatures and could be used as biomarkers for the detection of severe diseases such as cancer, as well as monitoring the therapeutic response. In this work, two kinds of MXene materials (Ti3C2 and Ti2C) were successfully synthesized and employed in the isolation of EVs from the biological samples by taking advantage of the affinity interaction between the titanium (Ti) in MXenes and the phospholipid membrane of EVs. Compared with Ti2C MXene materials, TiO2 beads and the other EVs isolation methods, Ti3C2 MXene materials exhibited excellent isolation performance via the coprecipitation with EVs due to the abundant unsaturated coordination of Ti2+/Ti3+, and the dosage of materials was the lowest. Meanwhile, the whole isolation process could be done within 30 min and integrated well with the following analysis of proteins and ribonucleic acids (RNAs), which was also convenient and economic. Furthermore, the Ti3C2 MXene materials were used to isolate the EVs from the blood plasma of colorectal cancer (CRC) patients and healthy donors. Proteomics analysis of EVs showed that 67 proteins were up-regulated, in which most of them were closely related to CRC progression. These findings indicate that the MXene material-based EVs isolation method via coprecipitation provides an efficient tool for early diagnosis of diseases.

Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves.

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-assisted laser desorption/ionization-mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was used to produce expression profiles of 8846 cells, with a median of 2352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in T. mairei leaves. Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell-type annotation, most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells; phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells (including the stomatal complex and guard cells); and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT_2, and bHLH46. Our research establishes the transcriptional landscape of major cell types in T. mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.

[Determination of seven monoaromatic hydrocarbon metabolites by ultra performance liquid chromatography-tandem mass spectrometry].

Monoaromatic hydrocarbons (MAHs) such as benzene, toluene, and xylene are important anthropogenic pollutants in the urban atmosphere. The detection of urinary MAH metabolites are included in human biomonitoring programs in several countries, including Canada, the United States, Italy, and Germany, because their evaluation is vital to monitor the exposure of humans to MAHs. To this end, herein, a method was developed for the determination of seven MAH metabolites through ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). An aliquot of 0.5 mL urine was fortified with an isotopic labeled internal standard solution before being hydrolyzed by 40 µL of 6 mol/L HCl solution, followed by extraction using a 96-well EVOLUTE®EXPRESS ABN solid-phase extraction plate. The samples were washed with 1.0 mL of methanol-water (10∶90, v/v) and eluted with 1.0 mL methanol. The eluate was diluted four times with water prior to use in instrumental analysis. Chromatographic separation was achieved using an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 µm), with gradient elution using 0.1% formic acid as mobile phase A and methanol as mobile phase B. The detection of seven analytes was performed using a triple-quadrupole mass spectrometer equipped with a negative electrospray ionization source in the multiple reaction monitoring mode. The linear ranges of the seven analytes varied from 0.1-20 µg/L to 2.5-500 mg/L, with correlation coefficients greater than 0.995. The method detection limits were 1.5, 0.02, 0.1, 900, 0.6, and 4 µg/L for trans,trans-muconic acid (MU), S-phenylmercapturic acid (PMA), S-benzylmercapturic acid (BMA), hippuric acid (HA), 2-methyl hippuric acid (2MHA), and 3-methyl hippuric acid (3MHA)+4-methyl hippuric acid (4MHA), respectively. The limits of quantification were 5, 0.05, 0.4, 3000, 2, and 12 µg/L for MU, PMA, BMA, HA, 2MHA, and 3MHA+4MHA, respectively. The method was verified by spiking urine samples at three different concentration levels, with recovery rates ranging from 84% to 123%. The intra- and inter-day precisions were 1.8%-8.6% and 1.9%-21.4%, respectively. The extraction efficiencies were 68%-99%, and the matrix effects ranged from -11% to -87%. The urine samples obtained from the German external quality assessment scheme (round 65) were used to assess the accuracy of this method. Both high and low concentrations of MU, PMA, HA, and methyl hippuric acid were within the tolerance range. All analytes in the urine samples were found to be stable for up to seven days at room temperature (20 ℃, absence of light), with less than 15% change in concentration. Analytes in urine samples were found to be stable for at least 42 d at 4 ℃ and -20 ℃, or for six freeze-thaw cycles and up to 72 h in an autosampler (8 ℃). The method was applied to the analysis of 16 non-smokers' and 16 smokers' urine samples. The detection rates of MU, BMA, HA, and 2MHA were 100% in both non-smokers' and smokers' urine samples. PMA was detected in 75% non-smokers' and 100% smokers' urine samples. 3MHA+4MHA was detected in 81% non-smokers' urine and in all smokers' urine samples. Statistical differences were found for MU, PMA, 2MHA, and 3MHA+4MHA between the two groups (p<0.001). The established method has good robustness and can provide reliable results. The experiments were carried out in a high-throughput manner with large sample sizes, owing to the small sample volume, and allowed the successful detection of the seven MAH metabolites in human urine.

Potential unreliability of ALK variant allele frequency in the efficacy prediction of targeted therapy in NSCLC.

Anaplastic lymphoma kinase (ALK) is the most common fusion gene involved in non-small cell lung cancer (NSCLC), and remarkable response has been achieved with the use of ALK tyrosine kinase inhibitors (ALK-TKIs). However, the clinical efficacy is highly variable. Pre-existing intratumoral heterogeneity (ITH) has been proven to contribute to the poor treatment response and the resistance to targeted therapies. In this work, we investigated whether the variant allele frequencies (VAFs) of ALK fusions can help assess ITH and predict targeted therapy efficacy. Through the application of next-generation sequencing (NGS), 7.2% (326/4548) of patients were detected to be ALK positive. On the basis of the adjusted VAF (adjVAF, VAF normalization for tumor purity) of four different threshold values (adjVAF < 50%, 40%, 30%, or 20%), the association of ALK subclonality with crizotinib efficacy was assessed. Nonetheless, no statistical association was observed between median progression-free survival (PFS) and ALK subclonality assessed by adjVAF, and a poor correlation of adjVAF with PFS was found among the 85 patients who received first-line crizotinib. Results suggest that the ALK VAF determined by hybrid capture-based NGS is probably unreliable for ITH assessment and targeted therapy efficacy prediction in NSCLC.

Ultra-rapid Idylla™ EGFR mutation screening followed by next-generation sequencing: An integrated solution to molecular diagnosis of non-small cell lung cancer.

Background: Rapid profiling of the EGFR mutations is crucial to help clinicians choose the optimal treatment for patients with advanced/metastatic Non-Small Cell Lung Cancer (NSCLC). Unfortunately, current diagnostic techniques, including ARMS-PCR and NGS, generally require several days to deliver final results. This diagnostic delay may lead to treatment delays for patients who are worsening rapidly. Methods: This study introduced the ultra-rapid Idylla™ system for rapid, sensitive and specific identification of the EGFR mutations among Chinese NSCLC patients. Idylla™ EGFR Assay, an integrated cartridge running on the Idylla™ system, which can detect 51 EGFR mutations directly from Formalin-Fixed, Paraffin-Embedded (FFPE) samples within 2.5 hours, was used in this study. The sensitivity and specificity of the Idylla™ system were evaluated in comparison with ARMS-PCR or NGS using 95 clinical samples. Results: The Idylla™ system achieved a sensitivity of 97.6%, a specificity of 100%, and an overall concordance of 97.9% for 95 retrospective samples. When compared to ARMS-PCR, the Idylla™ system demonstrated high accuracy with an overall agreement of 97.1% (34/35), a sensitivity of 95.2% (20/21) (95% CI, 76.2% - 99.9%), and an estimated specificity of 100% (12/12) (95% CI, 76.8% - 100%) for 35 prospective samples. Conclusions: This Idylla system provides a rapid, accurate and simple approach for screening EGFR mutations, which can guide Tyrosine Kinase Inhibitors (TKI) treatment for NSCLC patients in a timely manner.

Loss of NLRP3 reduces oxidative stress and polarizes intratumor macrophages to attenuate immune attack on endometrial cancer.

Introduction: The interaction between endometrial cancer (EMC) cells and intratumoral macrophages plays a significant role in the development of the disease. PYD domains-containing protein 3 (NLRP3) inflammasome formation triggers caspase-1/IL-1ß signaling pathways and produces reactive oxygen species (ROS) in macrophages. However, the role of NLRP3-regulated ROS production in macrophage polarization and the subsequent growth and metastasis of EMC remains unknown. Methods: We conducted bioinformatic analysis to compare NLRP3 levels in intratumoral macrophages from EMC and normal endometrium. In vitro experiments involved knocking out NLRP3 in macrophages to shift the polarization from an anti-inflammatory M1-like phenotype to a proinflammatory M2-like phenotype and reduce ROS production. The impact of NLRP3 depletion on the growth, invasion, and metastasis of co-cultured EMC cells was assessed. We also evaluated the effect of NLRP3 depletion in macrophages on the growth and metastasis of implanted EMC cells in mice. Results: Our bioinformatic analysis showed significantly lower NLRP3 levels in intratumoral macrophages from EMC than those from normal endometrium. Knocking out NLRP3 in macrophages shifted their polarization to a proinflammatory M2-like phenotype and significantly reduced ROS production. NLRP3 depletion in M2-polarized macrophages increased the growth, invasion, and metastasis of co-cultured EMC cells. NLRP3 depletion in M1-polarized macrophages reduced phagocytic potential, which resulted in weakened immune defense against EMC. Additionally, NLRP3 depletion in macrophages significantly increased the growth and metastasis of implanted EMC cells in mice, likely due to compromised phagocytosis by macrophages and a reduction in cytotoxic CD8+ T cells. Discussion: Our results suggest that NLRP3 plays a significant role in regulating macrophage polarization, oxidative stress, and immune response against EMC. NLRP3 depletion alters the polarization of intratumoral macrophages, leading to weakened immune defense against EMC cells. The reduction in ROS production by the loss of NLRP3 may have implications for the development of novel treatment strategies for EMC.

MYD88L265P and MYD88other variants show different molecular characteristics and prognostic significance in diffuse large B-cell lymphoma.

PURPOSE: This study aims to investigate the clinical and molecular differences between diffuse large B-cell lymphoma (DLBCL) patients with MYD88L265P and MYD88other. METHODS: DLBCL patients with MYD88 variations were collected from the Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CHCAMS), and Suzhou Municipal Hospital from February 6th, 2007 to May 20th, 2022. Clinicopathological parameters and treatment outcomes between MYD88L265P and MYD88other were investigated. RESULTS: A total of 132 patients with MYD88 variations from a cohort of 475 DLBCL patients were included, among which, 78 were MYD88L265P, while 54 were MYD88other. MYD88L265P was more common in non-GCB subtype than MYD88other (83% vs. 60%, P = 0.004). Besides, MYD88L265P was significantly related to higher proportion of testicle/ central nervous system involvement (31% vs. 6%, P < 0.001), PIM1 mutation (71% vs. 39%, P < 0.001), and PIM1 hypermutation (28% vs. 11%, P = 0.018), compared with MYD88other. Compared with MYD88L265P, MYD88other were more likely to have higher percentage of advanced stage (60% vs. 42%, P = 0.044), extranodal site ≥ 2 (45% vs. 28%, P = 0.044), elevated LDH (55% vs. 35%, P = 0.033), positive CD10 expression (36% vs. 16%, P = 0.009), BCL-6 translocation (20% vs. 8%, P = 0.033), and NOTCH pathway gene alteration (24% vs. 13%, P = 0.040). In non-GCB DLBCL subtype, patients with MYD88other were significantly associated with worse progression free survival (PFS) than those with MYD88L265P when treated initially with R-CHOP/R-CHOP-like regimen (P = 0.010). CONCLUSION: The findings of this study indicate that DLBCL patients with MYD88L265P and MYD88other are likely to be two subgroups with different clinical and molecular characteristics. The survival of patients with MYD88other is not superior than those with MYD88L265P, even poorer when focusing on the non-GCB subtype.

Detection of MET Polysomy by Next-generation Sequencing and Its Clinical Relevance for MET Inhibitors.

Next-generation sequencing (NGS) has failed to detect mesenchymal epithelial transition factor gene (MET) polysomy in previous studies. We included three non-small cell lung cancer (NSCLC) cohorts in this retrospective study to establish new criteria for detecting MET polysomy and to explore the clinical relevance of MET polysomy. Cohort 1 included 53 patients whose tissues were available for both FISH and NGS assays. Paired plasma and tissue samples were obtained from 261 patients with NSCLC as cohort 2. Cohort 3 included 46 patients with metastatic NSCLC, who presented with MET copy-number gain assessed by NGS. ROC analysis demonstrated that a cut-off point of 2.3 copies achieved the maximum Youden index in discriminating polysomy from normal copy number. Compared with the FISH test for MET polysomy, the sensitivity, specificity, and agreement of NGS were 90%, 90%, and 96.2%, respectively. Following optimization using maximum somatic allele frequency, the sensitivity and specificity of NGS for defining polysomy using plasma samples according to different circulating tumor DNA mutation frequencies were 42% and 63%. The concordance rate between tissue and plasma samples for detecting polysomy was 85%. Regarding the response to MET inhibitor, the median progression-free survival (PFS) of the MET amplification group was significantly higher than that of the polysomy group. The median PFS was similar between the polysomy and normal groups. Our results indicated that NGS may serve as an alternative method for detecting MET polysomy in NSCLC tissues. Moreover, patients with MET polysomy may not benefit from MET inhibitors. Significance: In this study, we established a methodology to differentiate polysomy from normal copy numbers and amplification using NGS. Moreover, this study suggests that it is critical to discriminate MET polysomy from amplification, for the former may dilute the clinical benefit of MET inhibitors.

Rotational stability of plate-haptic toric intraocular lenses in Asian eyes: risk period for intraocular lens rotation and its influencing factors.

PURPOSE: To investigate the rotational stability of plate-haptic toric intraocular lenses (IOLs) during 3-month follow-up. SETTING: Eye and ENT Hospital of Fudan University, Shanghai, China. DESIGN: Prospective observational study. METHODS: Patients with cataracts implanted with AT TORBI 709M toric IOLs were enrolled and followed at 1 hour, 1 day, 3 days, 1 week, 2 weeks, 1 month, and 3 months postoperatively. A linear mixed model of repeated measures was applied to investigate the time course of absolute IOL rotation change. The 2-week overall IOL rotation was analyzed in the age, sex, axial length (AL), lens thickness (LT), preexisting astigmatism, and white-to-white subgroups. RESULTS: A total of 328 eyes of 258 patients were included. The rotation from the end of surgery to 1 hour and 1 day to 3 days was significantly smaller compared with the rotation from 1 hour to 1 day but more than that at other time intervals in the overall group. 2 weeks postoperatively, the mean uncorrected distance visual acuity and remaining positive cylinder were 0.19 ± 0.22 logMAR and 0.60 ± 0.44 diopters, respectively. Significant between-group differences in 2-week overall rotation were found in the age, AL, and LT subgroups. CONCLUSIONS: Maximum rotation occurred within 1 hour to 1 day postoperatively, and the first 3 days postoperatively was a high-risk period for the plate-haptic toric IOL rotation. Surgeons should make the patients aware of this.

[Determination of phenoxyacetic herbicides, metabolites of organophosphorus and pyrethroid pesticides in human urine using solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry].

Pesticides are widely used in most agricultural areas to protect food crops but adversely affect ecosystems and human beings. Pesticides have attracted great public concern due to their toxic properties and ubiquitous occurrence in the environment. China is one of the largest users and producers of pesticides globally. However, limited data are available on pesticide exposure in humans, which warrants a method for quantification of pesticides in human samples. In the present study, we validated and developed a comprehensive and sensitive method for the quantification of two phenoxyacetic herbicides, two metabolites of organophosphorus pesticides and four metabolites of pyrethroid pesticides in human urine using 96-well plate solid phase extraction (SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). For this purpose, a systematic optimization of the chromatographic separation conditions and MS/MS parameters was conducted. Six solvents were optimized for the extraction and clean-up of human urine samples. The targeted compounds in the human urine samples were well separated within 16 min in one analytical run. A 1 mL aliquot of human urine sample was mixed with 0.5 mL sodium acetate buffer (0.2 mol/L) and hydrolyzed by ß-glucuronidase enzyme at 37 ℃ overnight. The eight targeted analytes were extracted and cleaned using an Oasis HLB 96-well solid phase plate and eluted with methanol. The separation of the eight target analytes was performed on a UPLC Acquity BEH C18 column (150 mm×2.1 mm, 1.7 µm) with gradient elution using 0.1% (v/v) acetic acid in acetonitrile and 0.1% (v/v) acetic acid in water. The analytes were identified using the multiple reaction monitoring (MRM) mode under negative electrospray ionization (ESI-) and quantified by isotope-labelled analogs. Para-nitrophenol (PNP), 3,5,6-tricholor-2-pyridinol (TCPY) and cis-dichlorovinyl-dimethylcyclopropane carboxylic acid (cis-DCCA) exhibited good linearities ranging from 0.2 to 100 µg/L, and 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxy benzoic acid (4F-3PBA), 2,4-dicholorphenoxyacetic acid (2,4-D), trans-dichlorovinyl-dimethylcyclopropane carboxylic acid (trans-DCCA) and 2,4,5-tricholorphenoxyacetic acid (2,4,5-T) showed linearity ranging from 0.1 to 100 µg/L with correlation coefficients all above 0.9993. Method detection limits (MDLs) and method quantification limits (MQLs) of targeted compounds were in the range of 0.02 to 0.07 µg/L and 0.08 to 0.2 µg/L, respectively. The spiked recoveries of target compounds at three levels of 0.5, 5 and 40 µg/L were 91.1% to 110.5%. The inter- and intra-day precisions of targeted analytes were 2.9% to 7.8% and 6.2% to 10%, respectively. This method was applied to the analysis of 214 human urine samples across China. The results showed that all the targeted analytes, except 2,4,5-T, were detected in human urine. The detection rates of TCPY, PNP, 3-PBA, 4F-3PBA, trans-DCCA, cis-DCCA, and 2,4-D were 98.1%, 99.1%, 94.4%, 2.80%, 99.1%, 63.1% and 94.4%, respectively. The median concentration of targeted analytes in a decreasing order were: 2.0 µg/L (TCPY), 1.8 µg/L (PNP), 0.99 µg/L (trans-DCCA), 0.81 µg/L (3-PBA), 0.44 µg/L (cis-DCCA), 0.35 µg/L (2,4-D) and below MDLs (4F-3PBA ). For the first time, we developed a method to extract and purify specific biomarkers of pesticides from human samples based on offline 96-well SPE. This method has the advantages of simple operation, high sensitivity, and high accuracy. Moreover, up to 96 human urine samples were analyzed in one batch. It is suitable for the determination of eight specific pesticides and their metabolites in large sample sizes.

A multiscale functional map of somatic mutations in cancer integrating protein structure and network topology.

A major goal of cancer biology is to understand the mechanisms underlying tumorigenesis driven by somatically acquired mutations. Existing computational approaches focus on either scoring the pathogenicity of mutations or characterizing their effects at specific scales. Here, we established a unified computational framework, NetFlow3D, that systematically maps the multiscale mechanistic effects of somatic mutations in cancer. The establishment of NetFlow3D hinges upon the Human Protein Structurome, a complete repository we first compiled that incorporates the 3D structures of every single protein as well as the binding interfaces for all known PPIs in humans. The vast majority of 3D structural information was resolved by recent deep learning algorithms. By applying NetFlow3D to 415,017 somatic protein-altering mutations in 5,950 TCGA tumors across 19 cancer types, we identified 1,656 intra- and 3,343 inter-protein 3D clusters of mutations throughout the Human Protein Structurome, of which ~50% would not have been found if using only experimentally-determined protein structures. These 3D clusters have converging effects on 377 cellular subnetworks. Compared to canonical PPI network analyses, NetFlow3D achieved a 5.5-fold higher statistical power for identifying significantly dysregulated subnetworks. The majority of identified subnetworks were previously obscured by the overwhelming background noise of non-clustered passenger mutations, including portions of non-canonical PRC1, mediator complex, MCM2-7 complex, neddylation of cullins, complement system, TRiC, etc. NetFlow3D and our pan-cancer results can be accessed from http://netflow3d.yulab.org. This work shows that mapping how individual mutations act across scales requires the integration of their local spatial organization on protein structures and their global topological organization in the PPI network.