Serum NfL and EGFR/NfL ratio mRNAs as biomarkers for phenotype and disease severity of myelin oligodendrocyte glycoprotein IgG-associated disease.

Background and purpose: Myelin oligodendrocyte glycoprotein (MOG) IgG is frequently elevated in pediatric patients with acquired demyelinating syndrome (ADS). However, no specific biomarkers exist for phenotype classification, symptom severity, prognosis, and treatment guidance of MOG-IgG-associated disease (MOGAD). This study evaluated neurofilament light chain (NfL) and endothelial growth factor receptor (EGFR) mRNA expression levels in serum and cerebrospinal fluid (CSF) as potential biomarkers for MOGAD in Chinese children. Methods: This was a cross-sectional and single-center study. We enrolled 22 consecutive pediatric patients hospitalized with MOGAD and 20 control pediatric patients hospitalized for noninflammatory neurological diseases in Hebei Children's Hospital. Serum and CSF were collected from MOGAD patients within 3 days before immunotherapy. The mRNA levels of NfL and EGFR in serum and CSF were measured by real-time polymerase chain reaction (qPCR), and the EGFR/NfL ratio mRNA was calculated. These measurement values were then compared between disease groups and among MOGAD phenotypes. In addition, the correlations between the mRNAs of three markers (NfL, EGFR, EGFR/NfL ratio), extended disability status scale (EDSS) scores, and clinical phenotypes were analyzed. Results: Serum and CSF NfL mRNA levels were significantly higher of acute-stage MOGAD patients than those of control patients (p< 0.05 and p< 0.01, respectively), while the mRNA levels of serum EGFR and EGFR/NfL ratio were significantly lower of MOGAD patients than those of controls (p < 0.05, p < 0.0001). Serum NfL mRNA was significantly correlated with mRNA of serum EGFR (r =0.480, p < 0.05). Serum and CSF NfL mRNA levels in MOGAD patients with the ADEM-like phenotype were also significantly higher than those in control patients (p < 0.01, p < 0.01) and optic neuritis (ON) phenotype (p < 0.05, p < 0.05). Both mRNAs of NfL in CSF and EGFR/NfL ratio in serum were correlated with EDSS scores (p < 0.05, r = 0.424; p < 0.05, r= -0.521). Conclusion: The mRNA levels of elevated NfL in serum and CSF as well as lower EGFR and EGFR/NfL ratio in serum could help distinguish acute-phase MOGAD. Higher mRNA levels of NfL in serum and CSF of MOGAD patients help distinguish ADEM-like phenotype. In addition, serum EGFR/NfL mRNA ratio is indicative of disease severity in pediatric patients with MOGAD. Further investigations are warranted to elucidate the pathological mechanisms underlying these associations.

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.

[Influence of Serum TGF-ß1 and EGFR Levels on the Therapeutic Effect of High-Dose AraC in Patients with Acute Myeloid Leukemia Based on the Decision Curve].

OBJECTIVE: To analyze the influence of serum levels of transforming growth factor-ß1 (TGF-ß1) and epidermal growth factor receptor (EGFR) on the therapeutic effect of high-dose cytarabine (HD-AraC) in patients with acute myeloid leukemia (AML). METHODS: 98 patients with AML treated in our hospital from January 2019 to June 2020 were selected as the research subjects, all patients were treated with HD-AraC for 1 course of treatment every week. The effect of 2 groups were evaluated during after one course of treatment and divided into effective group and ineffective group, statistical table of baseline data was designed, the baseline data of 2 groups were counted in detail, the baseline data and serum levels of TGF-ß1 and EGFR of 2 groups were compared, Logistic regression analysis was used to examine the relationship between the levels of serum TGF-ß1, EGFR and the therapeutic effect of HD-AraC in patients with AML, the value of serum TGF-ß1 and EGFR levels in predicting the therapeutic effect of HD-AraC in AML patients was analyzed based on ROC curve and decision curve. RESULTS: After 1 course of treatment, among the 98 patients, 26 cases had complete remission, 38 cases had partially remission and 34 cases no remission, the total effective rate was 65.31% (64/98); after comparing data of 2 groups, Logistic regression analysis showed that the overexpression of serum EGFR before treatment might be a risk factor for the ineffective treatment of HD-AraC in AML patients (OR>1, P<0.05), overexpression of serum TGF-ß1 before treatment might be a protective factor for the ineffective treatment of HD-AraC in AML patients (OR<1, P<0.05); the ROC curve results showed that the AUC of serum EGFR and TGF-ß1 before treatment in predicting the risk of ineffective HD-AraC treatment in AML patients were >0.70, which had certain predictive value. The decision curve results showed that in the threshold range of 0.15-044, the prediction model combined with serum EGFR and TGF-ß1 levels in predicting the net benefit rate of HD-AraC treatment in AML patients was better than that of serum EGFR or serum TGF-ß1 alone. CONCLUSION: The levels of serum TGF-ß1 and EGFR affect the therapeutic effect of HD-AraC in patients with AML and increase the risk of ineffective treatment, serum TGF-ß1 and EGFR can be used to predict the risk of ineffective HD-AraC treatment in AML patients, and the combined prediction of net benefit rate is higher.

Mutation status and postresection survival of patients with non-small cell lung cancer brain metastasis: implications of biomarker-driven therapy.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is the most common primary tumor to develop brain metastasis. Prognostic markers are needed to better determine survival after neurosurgical resection of intracranial disease. Given the importance of mutation subtyping in determining systemic therapy and overall prognosis of NSCLC, the authors examined the prognostic value of mutation status for postresection survival of patients with NSCLC brain metastasis. METHODS: The authors retrospectively analyzed all cases of NSCLC brain metastasis with available molecular testing data that were resected by a single surgeon at a single academic center from January 2009 to February 2019. Mutation status, demographic characteristics, clinical factors, and treatments were analyzed. Association between predictive variables and overall survival after neurosurgery was determined with Cox regression. RESULTS: Of the included patients (n = 84), 40% were male, 76% were smokers, the mean ± SD Karnofsky Performance Status was 85 ± 14, and the mean ± SD age at surgery was 63 ± 11 years. In total, 23%, 26%, and 4% of patients had EGFR, KRAS, and ALK/ROS1 alterations, respectively. On multivariate analysis, survival of patients with EGFR (HR 0.495, p = 0.0672) and KRAS (HR 1.380, p = 0.3617) mutations were not significantly different from survival of patients with wild-type (WT) tumor. However, the subgroup of patients with EGFR mutation who also received tyrosine kinase inhibitor (TKI) therapy had significantly prolonged survival (HR 0.421, p = 0.0471). In addition, postoperative stereotactic radiosurgery (HR 0.409, p = 0.0177) and resected tumor diameter < 3 cm (HR 0.431, p = 0.0146) were also significantly associated with prolonged survival, but Graded Prognostic Assessment score ≤ 1.0 (HR 2.269, p = 0.0364) was significantly associated with shortened survival. CONCLUSIONS: Patients with EGFR mutation who receive TKI therapy may have better survival after resection of brain metastasis than patients with WT tumor. These results may inform counseling and decision-making regarding the appropriateness of resection of NSCLC brain metastasis.

Influence of Serum TGF-β1 and EGFR Levels on the Therapeutic Effect of High-Dose AraC in Patients with Acute Myeloid Leukemia Based on the Decision Curve / 中国实验血液学杂志

OBJECTIVE@#To analyze the influence of serum levels of transforming growth factor-β1 (TGF-β1) and epidermal growth factor receptor (EGFR) on the therapeutic effect of high-dose cytarabine (HD-AraC) in patients with acute myeloid leukemia (AML).@*METHODS@#98 patients with AML treated in our hospital from January 2019 to June 2020 were selected as the research subjects, all patients were treated with HD-AraC for 1 course of treatment every week. The effect of 2 groups were evaluated during after one course of treatment and divided into effective group and ineffective group, statistical table of baseline data was designed, the baseline data of 2 groups were counted in detail, the baseline data and serum levels of TGF-β1 and EGFR of 2 groups were compared, Logistic regression analysis was used to examine the relationship between the levels of serum TGF-β1, EGFR and the therapeutic effect of HD-AraC in patients with AML, the value of serum TGF-β1 and EGFR levels in predicting the therapeutic effect of HD-AraC in AML patients was analyzed based on ROC curve and decision curve.@*RESULTS@#After 1 course of treatment, among the 98 patients, 26 cases had complete remission, 38 cases had partially remission and 34 cases no remission, the total effective rate was 65.31% (64/98); after comparing data of 2 groups, Logistic regression analysis showed that the overexpression of serum EGFR before treatment might be a risk factor for the ineffective treatment of HD-AraC in AML patients (OR>1, P<0.05), overexpression of serum TGF-β1 before treatment might be a protective factor for the ineffective treatment of HD-AraC in AML patients (OR<1, P<0.05); the ROC curve results showed that the AUC of serum EGFR and TGF-β1 before treatment in predicting the risk of ineffective HD-AraC treatment in AML patients were >0.70, which had certain predictive value. The decision curve results showed that in the threshold range of 0.15-044, the prediction model combined with serum EGFR and TGF-β1 levels in predicting the net benefit rate of HD-AraC treatment in AML patients was better than that of serum EGFR or serum TGF-β1 alone.@*CONCLUSION@#The levels of serum TGF-β1 and EGFR affect the therapeutic effect of HD-AraC in patients with AML and increase the risk of ineffective treatment, serum TGF-β1 and EGFR can be used to predict the risk of ineffective HD-AraC treatment in AML patients, and the combined prediction of net benefit rate is higher.

Plasma Metabolome Profiling by High-Performance Chemical Isotope-Labelling LC-MS after Acute and Medium-Term Intervention with Golden Berry Fruit (Physalis peruviana L.), Confirming Its Impact on Insulin-Associated Signaling Pathways.

PURPOSE: Golden berry (Physalis peruviana L.) is an exotic fruit exported from Colombia to different countries around the world. A review of the literature tends to demonstrate a hypoglycaemic effect with an improvement in insulin sensitivity after oral ingestion of fruit extracts in animal models. However, little is known about their potential effects in humans, and very little is known about the mechanisms involved. This study aimed at identifying discriminant metabolites after acute and chronic intake of golden berry. METHOD: An untargeted metabolomics strategy using high-performance chemical isotope-labelling LC-MS was applied. The blood samples of eighteen healthy adults were analysed at baseline, at 6 h after the intake of 250 g of golden berry (acute intervention), and after 19 days of daily consumption of 150 g (medium-term intervention). RESULTS: Forty-nine and 36 discriminant metabolites were identified with high confidence, respectively, after the acute and medium-term interventions. Taking into account up- and downregulated metabolites, three biological networks mainly involving insulin, epidermal growth factor receptor (EGFR), and the phosphatidylinositol 3-kinase pathway (PI3K/Akt/mTOR) were identified. CONCLUSIONS: The biological intracellular networks identified are highly interconnected with the insulin signalling pathway, showing that berry intake may be associated with insulin signalling, which could reduce some risk factors related to metabolic syndrome. Primary registry of WHO.

Validation of a next-generation sequencing assay for the detection of EGFR mutations in cell-free circulating tumor DNA.

Detection of EGFR mutations from blood plasma represents a gentle, non-invasive alternative to rebiopsy and can therefore be used for therapy monitoring of non-small-cell lung cancer (NSCLC) patients. The aim of this project was to investigate whether the Reveal ctDNA™ 28 NGS assay (ArcherDX, Boulder, CO), has a comparable sensitivity and specificity to droplet digital PCR (ddPCR, gold-standard) and is therefore suitable for therapy monitoring of progressing lung cancer patients. First, we validated the NGS assay with a commercially available reference material (SeraCare, Massachusetts, US). Using an input of 22 ng, a sensitivity of 96% and a specificity of 100% could be achieved for variant allele frequencies (VAF) of 0.5%. For variants at a VAF of 0.1% the sensitivity was substantially reduced. Next, 28 plasma samples from 16 patients were analyzed and results were compared to existing ddPCR data. This comparative analysis of patient samples revealed a concordance of 91% between NGS and ddPCR. These results confirm that the Reveal ctDNA™ 28 NGS assay can be used for therapy monitoring of patients under TKI therapy. However, due to the slightly superior sensitivity of ddPCR, a combination of NGS (with broad coverage of a large number of genomic loci) and ddPCR (with targeted highly sensitive detection of specific mutations) might be the ideal approach.

A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies.

BACKGROUND: A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen® EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies. RESULTS: Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven-log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen® EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R2≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen® EGFR Liquid assay platform showed better sensitivity than the Therascreen® EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level. CONCLUSION: For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.

Impact of detecting plasma EGFR mutations with ultrasensitive liquid biopsy in outcomes of NSCLC patients treated with first- or second-generation EGFR-TKIs.

BACKGROUND: Few trials have evaluated the utility of liquid biopsies to detect epidermal growth factor receptor mutations (EGFRm) at the time of response evaluation and its association with the clinical characteristics and outcomes of non-small-cell lung cancer (NSCLC) patients. OBJECTIVE: This study aimed to evaluate, in a real-world clinical setting, the prevalence of plasma EGFRm and its association with the clinical characteristics, response and survival outcomes of NSCLC patients under treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). METHODS: This observational study enrolled advanced or metastatic NSCLC patients, with confirmed tumor EGFRm, receiving treatment with first- or second-generation EGFR-TKIs. Blood samples for the detection of plasma EGFRm were collected at the time of response evaluation and processed using the Target Selector™ assay. The main outcomes were the detection rate of plasma EGFRm, median Progression-Free Survival (PFS) and Overall Survival (OS) according to plasma EGFR mutational status. RESULTS: Of 84 patients, 50 (59.5%) had an EGFRm detected in plasma. After a median follow-up of 21.1 months, 63 patients (75%) had disease progression. The detection rate of plasma EGFRm was significantly higher in patients with disease progression than in patients with partial response or stable disease (68.3% versus 33.3%; P< 0.01). PFS and OS were significantly longer in patients without plasma EGFRm than among patients with plasma EGFRm (14.3 months [95% CI, 9.25-19.39] vs 11.0 months [95% CI, 8.61-13.46]; P= 0.034) and (67.8 months [95% CI, 39.80-95.94] vs 32.0 months [95% CI, 17.12-46.93]; P= 0.006), respectively. A positive finding in LB was associated with the presence of ⩾ 3 more metastatic sites (P= 0.028), elevated serum carcinoembryonic (CEA) at disease progression (P= 0.015), and an increase in CEA with respect to baseline levels (P= 0.038). CONCLUSIONS: In NSCLC patients receiving EGFR-TKIs, the detection of plasma EGFRm at the time of tumor response evaluation is associated with poor clinical outcomes.

Evaluation of the Idylla ctEGFR mutation assay to detect EGFR mutations in plasma from patients with non-small cell lung cancers.

The assessment of EGFR mutations is recommended for the management of patients with non-small cell lung cancer (NSCLC). Presence of EGFR mutation is associated with response or resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI). Liquid biopsy is nowadays widely used for the detection of resistance to EGFR-TKI. We evaluated here the performance of the Idylla ctEGFR mutation assay for the detection of EGFR mutations in circulating tumour DNA (ctDNA) in plasma from patients with NSCLC. Previously characterized plasma samples from 38 patients with NSCLC were analysed using 2 different analytical conditions (C1 and C2). The limit of detection (LOD) was evaluated using 2 mL of healthy donor plasma spiked with commercial DNA controls. Overall agreement, sensitivity and specificity were 92.1%, 86.7% and 95.7% for C1 condition respectively and 94.7%, 86.7% and 100% for C2 condition respectively. The T790M secondary resistance mutation was detected in two samples out of 3. The Idylla system was able to detect the exon 19 deletion from 6 copies/mL and up to 91 copies/mL for the G719S mutation. These results support that the Idylla ctEGFR mutation assay is a rapid option for the detection of EGFR hotspots mutations in plasma samples, however a particular attention is needed for its interpretation.

Analysis of circulating tumour DNA to identify patients with epidermal growth factor receptor-positive non-small cell lung cancer who might benefit from sequential tyrosine kinase inhibitor treatment.

BACKGROUND: Survival data support the use of first-line osimertinib as the standard of care for epidermal growth factor receptor (EGFR)-positive non-small cell lung cancer (NSCLC). However, it remains unclear whether upfront osimertinib is superior to sequential first- or second-generation tyrosine kinase inhibitors (TKIs) followed by osimertinib for all patients. It is impossible to predict which patients are at high risk of progression, and this constitutes a major limitation of the sequential TKI approach. PATIENTS AND METHODS: A total of 830 plasma samples from 228 patients with stage IV, EGFR-positive NSCLC who were treated with first-line TKIs were analysed by digital polymerase chain reaction (dPCR). RESULTS: The circulating tumour DNA (ctDNA) levels helped to identify patients with significantly improved survival rate, regardless of the treatment. Patients treated with first- or second-generation TKIs (N = 189) with EGFR mutations in plasma at a mutant allele frequency (MAF) <7% before treatment initiation (low-risk patients) or who were ctDNA negative after 3 or 6 months of treatment and with an MAF <7% at diagnosis (high responders) had two-thirds lower risk of death than patients in the opposite situation (adjusted hazard ratio [HR] = 0.38; 95% confidence interval [CI]: 0.23-0.64 and HR = 0.22; 95% CI: 0.12-0.42, respectively). The median overall survival (OS) for low-risk patients and high responders treated with first- or second-generation TKIs was 34.2 months and not reached, respectively, regardless of second-line treatment. There were no significant difference in OS between low-risk or high-responder patients treated upfront with osimertinib (N = 39) and those treated under a sequential approach with osimertinib (N = 60). Median OS was not reached in both cases. CONCLUSIONS: Pre-treatment ctDNA levels identify low-risk patients, who may benefit from sequential TKI treatment. Information regarding EGFR mutation clearance can help to improve patient selection.

Correlations between serum cetuximab and EGFR-related markers, and skin disorders in head and neck cancer patients.

PURPOSE: Cetuximab inhibits epidermal growth factor receptor (EGFR) signaling in cancer and skin cells, thereby inducing anti-cancer effects and skin disorders. The present study aimed to evaluate the relationships between serum cetuximab and EGFR-related markers, and adverse effects in head and neck cancer patients. METHODS: Thirty-four head and neck cancer patients receiving weekly intravenous cetuximab were enrolled. Serum cetuximab levels were determined just before dosing. Blood samples for determination of serum EGFR-related markers including soluble epidermal growth factor receptor (sEGFR) and interleukin-6 (IL-6) were obtained. The severities of skin disorders, their medications, and hypomagnesemia treatment were also assessed. RESULTS: Serum levels of cetuximab and sEGFR were negatively and positively correlated with that of IL-6, respectively. The serum cetuximab level was twofold higher in the patients with a grade 2-3 skin rash than with a grade 0-1 rash. The serum cetuximab cutoff value related to severe skin rash was 71 µg/mL (sensitivity, 59%; and specificity, 94%). The use of a strong topical corticosteroid for skin rash was also associated with a higher serum cetuximab level. Serum levels of sEGFR and IL-6 had no correlations with the skin disorder severities or their medications. Hypomagnesemia treatment using intravenous magnesium sulfate was not related to serum cetuximab and EGFR-related markers. CONCLUSIONS: Head and neck cancer patients with a higher serum IL-6 level tended to have a lower serum cetuximab level. Serum cetuximab had positive correlations to skin rash severity and its medication in the study population.

Modifications of IGF2 and EGFR plasma protein concentrations in NAFLD patients after bariatric surgery.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is strictly associated with the epidemic of obesity and is becoming the most prevalent liver disease worldwide. In severe obesity, bariatric surgery (BS) is the most effective treatment not only for obesity but also for the associated metabolic co-morbidities, NAFLD, among others. To date, noninvasive diagnostic/prognostic methods cannot evaluate hepatic improvements following surgery. OBJECTIVES: We aimed to measure plasma level of insulin-growth factor-2 protein (IGF2) and epithermal growth factor receptor (EGFR), and to assess their relationship with clinical and biochemical parameters during the 12 months follow-up. METHODS: Demographic, clinical-biochemical data, and plasma IGF2 and EGFR were measured in 69 patients preoperatively (T0) and 6 and 12 months (T6M and T12M, respectively) after BS. Liver biopsy was performed at T0. Relationships between IGF2, EGFR, and several biochemical parameters were performed using Pearson or Spearman correlation analysis. RESULTS: IGF2 plasma level increases during follow-up, passing from 2.5 (1.8-15.5) at baseline to 13.3 (8.6-19.1) at T12M, p < 0.001. Conversely, EGFR showed a not significant reduction. At T12M, the plasma level of both markers was comparable to those of lean subjects. The clinical-biochemical parameters (BMI, glycated hemoglobin, HOMA-IR) also return to the normal range at T12M. Correlation analysis demonstrated that IGF2 was significantly associated with total bilirubin, direct bilirubin, and albumin at T0 while with blood glucose, ALT, GGT, and AST/ALT ratio at T6M and T12M. CONCLUSIONS: IGF2 plasma levels increase after bariatric surgery, and these changes are associated with the modification of hepatic biochemical parameters, even if other clinic or metabolic improvements cannot be excluded.

An Analysis of Rate of Progression and Associated Risk Factors of Chronic Kidney Disease of Uncertain Etiology in Sri Lanka.

Chronic kidney disease of uncertain etiology (CKD-u) is an important public health issue in Sri Lanka and around the world, but published evidence of the progression of this disease is scanty. Our aim is to analyze the progression patterns and the associated risk factors of definite and probable CKD-u cases. This observational study was based on records of CKD-u cohort from 2005-14 at Girandurukotte, an endemic area for CKD-u in Uva Province, Sri Lanka. Data (rate of progression, survival, and risk factors) were analyzed using R statistical software. CKD-u cases (379) were included in analyses based on the adequacy of variables. Mean age was 53 years, male-to-female ratio of 2.5:1, and smoking were significant risk factors (P <0.10) for CKD-u progression. Males had 2.3 times hazard for CKD-u survival than females, and males who smoked had 2.57 times risk of CKD-u progression than nonsmoking males. Faster eGFR decline rate of >5 mL/min/1.73 m2/year have been identified in 25% of the sample (n = 100); this group is significantly younger than the slower progression group (mean age 46 years) and was at an early stage at the time of presentation (mean eGFR 76.02). CKD-u progression was not equal in all patients but faster in young people who presented at earlier stages. Continuous exposure to environmental risk factors may influence the rate of progression. Females have higher CKD-u survival rates than males. Tobacco smoking was associated with a lower survival of CKD-u but could be a proxy indicative of other exposures.

Construction of a reference material panel for detecting KRAS/NRAS/EGFR/BRAF/MET mutations in plasma ctDNA.

BACKGROUND: The absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China. OBJECTIVE: This study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal-epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA). METHODS: Mutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel. RESULTS: 20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation. CONCLUSION: RM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.

EGFR-rich extracellular vesicles derived from highly metastatic nasopharyngeal carcinoma cells accelerate tumour metastasis through PI3K/AKT pathway-suppressed ROS.

Nasopharyngeal carcinoma (NPC) is the most common cancer with high metastatic potential that occurs in the epithelial cells of the nasopharynx. Distant metastases are the primary cause for treatment failure and mortality of NPC patients. However, the underlying mechanism responsible for the initiation of tumour cell dissemination and tumour metastasis in NPC is not well understood. Here, we demonstrated that epidermal growth factor receptor (EGFR) was highly expressed in tumour tissues of NPC patients with distant metastases and was associated with a decrease in reactive oxygen species (ROS). We also revealed that extracellular vesicles (EVs) transfer occurred from highly to poorly metastatic NPC cells, mediating cell-cell communication and enhancing the metastatic potential of poorly metastatic NPC cells. Further experiments indicated that EVs derived from highly metastatic NPC cells induced the up-regulation of EGFR and down-regulation of ROS in low metastatic NPC cells. Mechanistically, EGFR-rich EVs-mediated EGFR overexpression down-regulated intracellular ROS levels through the PI3K/AKT pathway, thus promoting the metastatic potential of poorly metastatic NPC cells. Strikingly, treatment with EVs secreted from highly metastatic NPC cells was significantly associated with rapid NPC progression and shorter survival in xenografted mice. These findings not only improve our understanding of EVs-mediated NPC metastatic mechanism but also have important implications for the detection and treatment of NPC patients accompanied by aberrant EGFR-rich EVs transmission.

Improved EGFR mutation detection sensitivity after enrichment by Cas9/sgRNA digestion and PCR amplification.

The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.

Measurement of Serum EGFR mRNA Expression is a Reliable Predictor of Treatment Response and Survival Outcomes in Non- Small Cell Lung Cancer.

BACKGROUND: EGFR over-expression plays a key role in the development and progression of lung cancer. However, its status as a prognostic biomarker for survival outcomes is unclear. OBJECTIVES: To evaluate the prognostic utility of serum EGFR mRNA expression in Non-Small cell lung cancer (NSCLC) for treatment response and survival. METHODS: EGFR mRNA levels were determined in serum using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Based on ROC curve, a cut off value of 16.0-fold increase was selected to categorize patients into low EGFR (≤ 16.0) and high EGFR (> 16.0) groups. RESULTS: A total of 350 subjects were included (78.3% males), with mean (± SD) age of 57.1 (± 11.2) years, and including 247 (70.6%) adenocarcinoma (ADC). Majority (73.1%) had metastatic (stage IV) disease. Patients had higher pre-treatment serum EGFR mRNA levels than controls [median fold-increase (min, max), 16.2 (1.9, 66.7). Serum EGFR mRNA levels significantly reduced in those who achieved objective response and disease control. Significantly longer OS and PFS was observed in subjects having baseline EGFR mRNA expression ≤ 16.0 fold- increase compared to those with > 16.0 fold- increase [median (95% CI) OS: 25.0 (14.9, NR) versus 7.7 (6.3, 8.9) months; HR (95% CI) 2.9 (2.3, 4.0), p < 0.001; and PFS: 9.9 (7.1, 11.5) versus 6.0 (4.1, 7.5) months; HR (95% CI) 1.8 (1.3, 2.4), p < 0.001]. CONCLUSION: Serum EGFR mRNA expression is a useful parameter for predicting treatment response and survival outcomes in NSCLC.

Prognostic impact of serum levels of EGFR and EGFR ligands in early-stage breast cancer.

Epidermal growth factor receptor (EGFR) and its ligands are involved in cancer pathogenesis. The emerging role of treatments co-targeting the EGFR system in breast cancer has increased the need to identify companion biomarkers. The aim of this study is to investigate whether pretreatment serum levels of EGFR and EGFR ligands in early-stage breast cancer patients might provide prognostic information as a stepping stone for further investigation. The study, which included 311 early-stage breast cancer patients, investigated associations between preoperative serum levels of EGFR and EGFR ligands (epidermal growth factor, heparin-binding epidermal growth factor (HBEGF), amphiregulin, transforming growth factor-α and betacellulin) and survival. Cutoffs were determined using Youden's method, and overall survival (OS) and invasive disease-free survival (IDFS) were evaluated using Cox regression. Preoperative S-EGFR < 60.3 ng/mL was associated with shorter OS and IDFS in both univariate analyses and when adjusting for standard prognostic factors (p < 0.05). Preoperative S-HBEGF < 21.4 pg/mL was associated with shorter OS in both univariate and multivariate analyses, whereas association with shorter IDFS could only be demonstrated in the univariate analysis. In conclusion, our study demonstrated shorter survival in early-stage breast cancer patients who had low pretreatment levels of either S-EGFR or S-HBEGF.

Detection of epidermal growth factor receptor mutations in peripheral blood circulating tumor DNA in patients with advanced non-small cell lung cancer: A PRISMA-compliant meta-analysis and systematic review.

BACKGROUND: The epidermal growth factor receptor (EGFR) mutation status related to the treatment approach for advanced non-small cell lung cancer (NSCLC) patients. This study aimed to evaluate the diagnostic accuracy of peripheral blood circulating tumor DNA (ctDNA) in EGFR mutated advanced NSCLC patients. METHOD: The related database was systematically searched with keywords until January 19, 2020. Studies contained the histopathological and cytological advanced NSCLC samples were included, and the diagnostic data were recorded for calculating sensitivity and specificity. I statistics were used for detecting heterogeneity across studies, and the meta-regression was performed to seek the source of heterogeneity. RESULT: A total of 32 studies with 4527 advanced NSCLC patients were included in our meta-analysis. Among them, 87% of the patients were diagnosed as stage IV. The pooled sensitivity of peripheral blood ctDNA was 0.70 (95% CI: 0.63-0.75, I = 81.76) and the pooled specificity was 0.98 (95% CI: 0.96-0.99, I = 88.33). The meta-regression showed that the prospective study design and the ARMS detection method were the main source of heterogeneity for sensitivity (P < .05), and the publication country (Asia or non-Asia) was the main source of heterogeneity for specificity (P < .01). CONCLUSION: ctDNA biopsy has high specificity and diagnostic accuracy in detection of EGFR mutation in advanced NSCLC patients. When the ctDNA gene test result is negative, we should fully consider the risk of missed diagnosis, and further tissue biopsy is still needed to undertake.