Electrospun EU/HPMC nanofibers decorated by ZIF-8 nanoparticle as the advanced electrochemical biosensor modifier for sensitive and selective detection of c-MET cancer biomarker in human plasma sample.

This research presents a selective and sensitive electrochemical biosensor for the detection of the mesenchymal-epithelial transition factor (c-MET). The biosensing is based on a modification of the SPCE (screen-printed carbon electrode) with the electrospun nanofiber containing eudragit (EU), hydroxypropyl methylcellulose (HPMC), and Zeolite imidazolate frameworks (ZIF-8) nanoparticles. EU/HPMC/ZIF-8 nanofibers have presented a high capability of electron transfer, and more active surface area than bare SPCE due to synergistic effects between EU, HPMC, and ZIF-8. On the other hand, EU/HPMC nanofibers provided high porosity, flexible structures, high specific surface area, and good mechanical strength. The presence of ZIF-8 nanoparticles improved the immobilization of anti-c-MET on the modified SPCE and also resulted in increasing the conductivity. By c-MET incubation on the modified SPCE, c-MET was connected to anti-c-MET, and consequently the electrochemical signal of [Fe(CN)6]3-/4- as the anion redox probe was reduced. In order to investigate the structural and morphological characteristics and elemental composition of electrospun nanofibers, various characterization methods including FE-SEM, XRD, FTIR, and EDS were used. Under optimum conditions with a working potential range -0.3-0.6 V (vs. Ag/AgCl), linear range (LR), correlation coefficient (R2), sensitivity, and limit of detection (LOD) were acquired at 100 fg/mL-100 ng/mL, 0.9985, 53.28 µA/cm2.dec, and 1.28 fg/mL, respectively. Moreover, the mentioned biosensor was investigated in a human plasma sample to determine c-MET and showed ideal results including reproducibility, stability, and good selectivity against other proteins.

Proteomic identification of biomarkers in maternal plasma that predict the outcome of rescue cerclage for cervical insufficiency.

OBJECTIVE: We sought to identify plasma protein biomarkers that are predictive of the outcome of rescue cerclage in patients with cervical insufficiency. METHODS: This retrospective cohort study included 39 singleton pregnant women undergoing rescue cerclage for cervical insufficiency (17-25 weeks) who gave plasma samples. Three sets of pooled plasma samples from controls (cerclage success, n = 10) and cases (cerclage failure, n = 10, defined as spontaneous preterm delivery at <33 weeks) were labeled with 6-plex tandem mass tag (TMT) reagents and analyzed by liquid chromatography-tandem mass spectrometry. Differentially expressed proteins between the two groups were selected from the TMT-based quantitative analysis. Multiple reaction monitoring-mass spectrometry (MRM-MS) analysis was further used to verify the candidate proteins of interest in patients with cervical insufficiency in the final cohort (n = 39). RESULTS: From MRM-MS analysis of the 40 proteins showing statistically significant changes (P < 0.05) from the TMT-based quantitative analysis, plasma IGFBP-2, PSG4, and PGLYRP2 levels were found to be significantly increased, whereas plasma MET and LXN levels were significantly decreased in women with cerclage failure. Of these, IGFBP-2, PSG4, and LXN levels in plasma were independent of cervical dilatation. A multiple-biomarker panel was developed for the prediction of cerclage failure, using a stepwise regression procedure, which included the plasma IGFBP-2, PSG4, and LXN (area under the curve [AUC] = 0.916). The AUC for this multiple-biomarker panel was significantly greater than the AUC for any single biomarker included in the multi-biomarker model. CONCLUSIONS: Proteomic analysis identified useful and independent plasma biomarkers (IGFBP-2, PSG4, and LXN; verified by MRM) that predict poor pregnancy outcome following rescue cerclage. Their combined analysis in a multi-biomarker panel significantly improved predictability.

The levels of soluble cMET ectodomain in the blood of patients with ovarian cancer are an independent prognostic biomarker.

The tyrosine kinase mesenchymal-epithelial transition (cMET) is typically overexpressed in up to 75% of patients with ovarian cancer, and cMET overexpression has been associated with poor prognosis. The proteolytic release of the soluble cMET (sMET) ectodomain by metalloproteases, a process called ectodomain shedding, reflects the malignant potential of tumour cells. sMET can be detected in the human circulation and has been proposed as biomarker in several cancers. However, the clinical relevance of sMET in ovarian cancer as blood-based biomarker is unknown and was therefore investigated in this study. sMET levels were determined by enzyme-linked immunosorbent assay in a set of 432 serum samples from 85 healthy controls and 86 patients with ovarian cancer (87% FIGO III/IV). Samples were collected at primary diagnosis, at four longitudinal follow-up time points during the course of treatment and at disease recurrence. Although there was no significant difference between median sMET levels at primary diagnosis of ovarian cancer vs. healthy controls, increased sMET levels at primary diagnosis were an independent predictor of shorter PFS (HR = 0.354, 95% CI: 0.130-0.968, P = 0.043) and shorter OS (HR = 0.217, 95% CI: 0.064-0.734, P = 0.014). In the follow-up samples, sMET levels were prognostically most informative after the first three cycles of chemotherapy, with high sMET levels being an independent predictor of shorter PFS (HR = 0.245, 95% CI: 0.100-0.602, P = 0.002). This is the first study to suggest that sMET levels in the blood can be used as an independent prognostic biomarker for ovarian cancer. Patients at high risk of recurrence and with poor prognosis, as identified based on sMET levels in the blood, could potentially benefit from cMET-directed therapies or other targeted regimes, such as PARP inhibitors or immunotherapy.

Inhibition of hepatocyte growth factor/c-Met signalling abrogates joint destruction by suppressing monocyte migration in rheumatoid arthritis.

OBJECTIVES: To determine the expression of hepatocyte growth factor (HGF) in RA biological fluids, the role of HGF in monocyte migration and the therapeutic effect of the c-Met inhibitor savolitinib in an arthritis model mice. METHODS: HGF/c-Met expression in serum, SF and synovial tissues (STs) obtained from RA patients and controls, as well as RA fibroblast-like synoviocytes (FLSs), was evaluated by ELISA and immunostaining. To determine the function of HGF in RA SF, we preincubated RA SF with a neutralizing anti-HGF antibody and measured the chemotactic ability of a human acute monocytic leukaemia cell line (THP-1). Additionally, examinations were conducted of SKG mice treated with savolitinib for 4 weeks. RESULTS: HGF levels in serum from RA patients were significantly higher than those in the controls and were decreased by drug treatment for 24 weeks. Additionally, the HGF level in SF from RA patients was higher than that in SF from OA patients. HGF and c-Met expression was also noted in RA STs. Stimulation of RA FLSs with TNF-α increased HGF/c-Met expression in a concentration-dependent manner, and c-Met signal inhibition suppressed production of fractalkine/CX3CL1 and macrophage inflammatory protein-1α/CCL3. When HGF was removed by immunoprecipitation, migration of THP-1 in RA SF was suppressed. In SKG mice, savolitinib significantly suppressed ankle bone destruction on µCT, with an associated reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. CONCLUSION: HGF produced by inflammation in synovium of RA patients activates monocyte migration to synovium and promotes bone destruction via a chemotactic effect and enhanced chemokine production.

MET somatic activating mutations are responsible for lymphovenous malformation and can be identified using cell-free DNA next generation sequencing liquid biopsy.

OBJECTIVE: Germline mutations of either the endothelial cell-specific tyrosine kinase receptor TIE2 or the glomulin (GLMN) gene are responsible for rare inherited venous malformations. Both genes affect the hepatocyte growth factor receptor c-Met, inducing vascular smooth muscle cell migration. Germline mutations of hepatocyte growth factor are responsible for lymphatic malformations, leading to lymphedema. The molecular alteration leading to the abnormal mixed vascular anomaly defined as lymphovenous malformation has remained unknown. METHODS: A group of 4 patients with lymphovenous malformations were selected. Plasma was obtained from both peripheral and efferent vein samples at the vascular malformation site for cell-free DNA extraction. When possible, we analyzed tissue biopsy samples from the vascular lesion. RESULTS: We have demonstrated that in all four patients, an activating MET mutation was present. In three of the four patients, the same pathogenic activating mutation, T1010I, was identified. The mutation was found at the tissue level for the patient with tissue samples available, confirming its causative role in the lymphovenous malformations. CONCLUSIONS: In the present study, we have demonstrated that cell-free DNA next generation sequencing liquid biopsy is able to identify the MET mutations in affected tissues. Although a wider cohort of patients is necessary to confirm its causative role in lymphovenous malformations, these data suggest that lymphovenous malformations could result from postzygotic somatic mutations in genes that are key regulators of lymphatic development. The noninvasiveness of the method avoids any risk of bleeding and can be easily performed in children. We are confident that the present pioneering results have provided a viable alternative in the future for lymphovenous malformation diagnosis, allowing for subsequent therapy tailored to the genetic defect.

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.

HGF-Induced PD-L1 Expression in Head and Neck Cancer: Preclinical and Clinical Findings.

Head and neck squamous cell carcinoma (HNSCC) is a widespread disease with a low survival rate and a high risk of recurrence. Nowadays, immune checkpoint inhibitor (ICI) treatment is approved for HNSCC as a first-line treatment in recurrent and metastatic disease. ICI treatment yields a clear survival benefit, but overall response rates are still unsatisfactory. As shown in different cancer models, hepatocyte growth factor/mesenchymal-epithelial transition (HGF/Met) signaling contributes to an immunosuppressive microenvironment. Therefore, we investigated the relationship between HGF and programmed cell death protein 1 (PD-L1) expression in HNSCC cell lines. The preclinical data show a robust PD-L1 induction upon HGF stimulation. Further analysis revealed that the HGF-mediated upregulation of PD-L1 is MAP kinase-dependent. We then hypothesized that serum levels of HGF and soluble programmed cell death protein 1 (sPD-L1) could be potential markers of ICI treatment failure. Thus, we determined serum levels of these proteins in 20 HNSCC patients before ICI treatment and correlated them with treatment outcomes. Importantly, the clinical data showed a positive correlation of both serum proteins (HGF and sPD-L1) in HNSCC patient's sera. Moreover, the serum concentration of sPD-L1 was significantly higher in ICI non-responsive patients. Our findings indicate a potential role for sPD-L1 as a prognostic marker for ICI treatment in HNSCC.

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients.

MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients (r = 0.57, p <10-10) was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients (P = 0.05; HR = 6.66). In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapy.

Phase I study of TAS-115, a novel oral multi-kinase inhibitor, in patients with advanced solid tumors.

TAS-115 is a novel MET, VEGFR, FMS and PDGFR inhibitor, developed to improve the continuity of drug administration with a relatively short half-life. We assessed its tolerability, safety, pharmacokinetics, efficacy, and pharmacodynamics in patients with solid tumors. This open-label, dose-escalation phase I study of TAS-115 consisted of three parts: part 1 (TAS-115 was administered orally once daily [SID]); part 2 and an expansion part (SID in a 5 days on/2 days off [5-on/2-off] schedule for 21 days per cycle). In part 1 (200-800 mg SID administered to 21 patients), systemic exposure after single administration increased almost dose-proportionally. Three dose-limiting toxicities (DLTs) were observed in three patients: grade 3 rash (650 mg), thrombocytopenia with bleeding, and rash (800 mg). The maximum tolerated dose (MTD) was determined as 650 mg SID. In part 2, the 5-on/2-off schedule was evaluated at the MTD to improve treatment exposure. No DLTs were observed and no patients required treatment interruption in cycle 1. During part 2 and the expansion part (N = 61), grade ≥3 treatment-related adverse events were reported in 47 patients, with neutropenia (24.6%), hypophosphatemia (21.3%), anemia, and thrombocytopenia (14.8% each), and leukocytopenia (11.5%) occurring in ≥10% of patients. The best overall response was stable disease in 31 of 82 patients (37.8%). An apparent reduction in fluorodesoxyglucose-uptake and bone scan index was observed in some patients. TAS-115 was generally well tolerated, with manageable toxicities and recommended phase II dose was estimated as 650 mg SID, 5-on/2-off. Furthermore, promising antitumor activity was observed.

Detection of serum protein and circulating mRNA of cMET, HGF EGF and EGFR levels in lung cancer patients to guide individualized therapy.

BACKGROUND: Reseptor tyrosine kinases (cMET and EGFR) are important in lung cancer targeted therapy. We believe if we can use them as markers for clinicians to help decide the diagnosis of lung cancer. This parameter will be important in serum samples of patients with lung cancer diagnosis and treatment. The aim of this study is aimed to evaluate the clinical utility of serum protein and circulating mRNA of cMET and HGF in lung cancer patients. We also analyzed the correlation of mRNA expression with clinicopathologic parameters. METHODS: We performed enzyme-linked immunosorbent assay (ELISA) to measure and compare serum protein and circulating mRNA of cMET and HGF levels in peripheral blood from 60 lung cancer patients and 40 healthy control group. RESULTS: We found that both protein and gene expression levels of serum c-MET, HGF and EGFR were significantly higher in patients with lung cancer than control group. There was no association between HGF, cMET, EGF, EGFR (both protein and gene) expression levels with age, gender, smoking habit, COPD, pathological types or tumor size, stage, metastatic-non metastatic adenocarcinoma-squamous carcinoma, SCLC-NSCLC. As a result of ROC analysis, serum cMET (AUC: 0.892) and HGF protein (AUC: 0.784) were diagnosed in lung cancer patients (Fig. 1). The AUC values of serum EGF and EGFR proteins were calculated to be 0.631 and 0.692, respectively. CONCLUSION: To our knowledge this is the first study comparing the levels of protein and mRNA in the serum material of HGF, c-MET, EGF and EGFR parameters in lung cancer patients' blood samples. Further prospective studies with more participants for better understanding of mechanism and effect for HGF and c-MET inhibitors in lung cancer will help us to identify of these biomarkers role for guiding us to sellect individualized itargeted therapies.

Phase I and Biomarker Study of Plerixafor and Bevacizumab in Recurrent High-Grade Glioma.

Purpose: Although antiangiogenic therapy for high-grade glioma (HGG) is promising, responses are not durable. Correlative clinical studies suggest that the SDF-1α/CXCR4 axis may mediate resistance to VEGFR inhibition. Preclinical data have demonstrated that plerixafor (a reversible CXCR4 inhibitor) could inhibit glioma progression after anti-VEGF pathway inhibition. We conducted a phase I study to determine the safety of plerixafor and bevacizumab in recurrent HGG.Patients and Methods: Part 1 enrolled 23 patients with a 3 × 3 dose escalation design to a maximum planned dose of plerixafor 320 µg/kg subcutaneously on days 1 to 21 and bevacizumab 10 mg/kg intravenously on days 1 and 15 of each 28-day cycle. Cerebrospinal fluid (CSF) and plasma samples were obtained for pharmacokinetic analyses. Plasma and cellular biomarkers were evaluated before and after treatment. Part 2 enrolled 3 patients and was a surgical study to determine plerixafor's penetration in tumor tissue.Results: In Part 1, no dose-limiting toxicities were seen at the maximum planned dose of plerixafor + bevacizumab. Treatment was well tolerated. After plerixafor 320 µg/kg treatment, the average CSF drug concentration was 26.8 ± 19.6 ng/mL. Plerixafor concentration in resected tumor tissue from patients pretreated with plerixafor was 10 to 12 µg/g. Circulating biomarker data indicated that plerixafor + bevacizumab induces rapid and persistent increases in plasma SDF-1α and placental growth factor. Progression-free survival correlated with pretreatment plasma soluble mesenchymal-epithelial transition receptor and sVEGFR1, and overall survival with the change during treatment in CD34+ progenitor/stem cells and CD8 T cells.Conclusions: Plerixafor + bevacizumab was well tolerated in HGG patients. Plerixafor distributed to both the CSF and brain tumor tissue, and treatment was associated with biomarker changes consistent with VEGF and CXCR4 inhibition. Clin Cancer Res; 24(19); 4643-9. ©2018 AACR.

A phase I study of LY3164530, a bispecific antibody targeting MET and EGFR, in patients with advanced or metastatic cancer.

PURPOSE: The phase I study characterized the safety, pharmacokinetics, anti-tumor activity, and recommended phase II dose/schedule of LY3164530 in patients with advanced or metastatic cancer. METHODS: Patients received LY3164530 on days 1 and 15 (Schedule 1: 300, 600, 1000, and 1250 mg) or Days 1, 8, 15, and 22 (Schedule 2: 500 and 600 mg) of each 28 days cycle. Dose escalation used a modified toxicity probability interval model. RESULTS: Dose escalation defined a maximum tolerated dose (MTD) of 1000 mg on Schedule 1 and 500 mg on Schedule 2. Treatment-emergent adverse events related to study treatment were consistent with epidermal growth factor receptor (EGFR) inhibition and included maculopapular rash/dermatitis acneiform (83%, Grade 3/4 17%), hypomagnesemia (55%, Grade 3/4 7%), paronychia (35%), fatigue (28%, Grade 3/4 3%), skin fissures (24%), and hypokalemia (21%, Grade 3/4 7%). Partial response was achieved in three patients on Schedule 2 with colorectal cancer (n = 2) or squamous cell cancer. Overall response rate (ORR) was 10.3%, disease control rate (ORR + stable disease [SD]) was 51.7 and 17.2% of patients had SD ≥ 4 months. The in vivo stability of the bispecific antibody was confirmed. Schedule 2 provided greater and more consistent inhibition of mesenchymal-epithelial transition (MET)/EGFR throughout the dosing interval than Schedule 1. CONCLUSIONS: Although this study defined the LY3164530 MTD and pharmacokinetics on both schedules, significant toxicities associated with EGFR inhibition and lack of a potential predictive biomarker limit future development. Nonetheless, the results provide insight into the development of bispecific antibody therapy.

Serum miR-658 induces metastasis of gastric cancer by activating PAX3-MET pathway: A population-based study.

BACKGROUND AND OBJECTIVE: MiR-658, paired box gene 3 (PAX3) and met proto-oncogene (MET) are overexpressed in gastric cancer while PAX3 and MET can be regulated by miRNA. Serum miR-658 may be associated with metastasis of gastric cancer (MGC) by affecting PAX3-MET pathway. METHODS: Ninety-eight gastric carcinoma patients with distant MGC (DM group) and ninety-six gastric carcinoma patients with no MGC (NM group) were recruited. Serum miR-658 was validated between DM and NM groups by using quantitative reverse transcription PCR (qRT-PCR). PAX3 and MET levels were measured by Western Blot. The molecular mechanism for the function of serum miR-658 was further validated in gastric cell lines. RESULTS: The results demonstrate that serum level of miR-658 is significantly lower in the NM group than in the DM group (P< 0.001). Meanwhile, the levels of PAX3 and MET are lower in the NM group than in the DM group too (P< 0.01). Both overexpression and silence of miR-658 significantly up-regulate or down-regulate the levels of PAX3 and MET in gastric cell lines (P< 0.05). CONCLUSIONS: The present findings demonstrate that elevated circulating miR-658 is associated with MGC by activating PAX3-MET pathway.

Predictive blood plasma biomarkers for EGFR inhibitor-induced skin rash.

Epidermal growth factor receptor overexpression in human cancer can be effectively targeted by drugs acting as specific inhibitors of the receptor, like erlotinib, gefitinib, cetuximab and panitumumab. A common adverse effect is a typical papulopustular acneiform rash, whose occurrence and severity are positively correlated with overall survival in several cancer types. We studied molecules involved in epidermal growth factor receptor signaling which are quantifiable in plasma, with the aim of identifying biomarkers for the severity of rash. With a predictive value for the rash these biomarkers may also have a prognostic value for survival and disease outcome.The concentrations of amphiregulin, hepatocyte growth factor (HGF) and calcidiol were determined by specific enzyme-linked immunosorbent assays in plasma samples from 211 patients.We observed a significant inverse correlation between the plasma concentration of HGF and overall survival in patients with an inhibitor-induced rash (p-value = 0.0075; mean overall survival low HGF: 299 days, high HGF: 240 days) but not in patients without rash. The concentration of HGF was also significantly inversely correlated with severity of rash (p-value = 0.00124).High levels of HGF lead to increased signaling via its receptor MET, which can activate numerous pathways which are normally also activated by epidermal growth factor receptor. Increased HGF/MET signaling might compensate the inhibitory effect of epidermal growth factor receptor inhibitors in skin as well as tumor cells, leading to less severe skin rash and decreased efficacy of the anti-tumor therapy, rendering the plasma concentration of HGF a candidate for predictive biomarkers.

[Correlation Research on HGF and c-Met of Chronic Erosive Gastritis Patients].

Objective To observe the correlation of hepatocyte growth factor (HGF) and Hepato- cyte growth factor receptor (c-Met ) in serum and gastric mucosa tissues of chronic erosive gastritis pa- tients. Methods Totally 70 patients with chronic erosive gastritis were selected and assigned to turbidity toxin intrinsic syndrome group and Gan-wei disharmony syndrome group, HGF expression level of ser- um,and HGF,c-Met expression level of gastric mucosa tissues were measured;the correlation of HGF and c-Met in gastric mucosa tissues, and the correlation of HGF in serum and gastric mucosa tissues were analyzed. Results The expression level of HGF and c-Met in turbidity toxin intrinsic syndrome group was higher than that in Gan-wei disharmony syndrome group (P <0. 05) ; the expression level of HGF in gastric mucosa tissues was positively correlated with c-Met(r =0. 831 , P <0. 05) ; the expression level of HGF in serum was positively correlated with that of gastric mucosa tissues(r =0. 656, P <0. 05). Conclusions There was correlation between turbidity toxin intrinsic syndrome of Chronic Erosive Gastri- tis patients and the expression level of HGF and c-Met.

High-MET status in non-small cell lung tumors correlates with receptor phosphorylation but not with the serum level of soluble form.

OBJECTIVES: The receptor tyrosine kinase MET is essential to embryonic development and organ regeneration. Its deregulation is associated with tumorigenesis. While MET gene amplification and mutations leading to MET self-activation concern only a few patients, a high MET level has been found in about half of the non-small cell lung cancers (NSCLCs) tested. How this affects MET activation in tumors is unclear. Also uncertain is the prognostic value, in cancer, of a phenomenon well described in cell models: MET shedding, i.e. its cleavage by membrane proteases leading to release of a soluble fragment into the medium. MATERIALS AND METHODS: A prospective cohort of 39 NSCLC patients was constituted at diagnosis or soon after. Normal tissues, tumor tissues, and blood samples were obtained. This allowed, for the same patient, synchronous determination of (i) the MET level in the tumor, (ii) receptor phosphorylation, and (iii) the concentration of soluble MET fragment (sMET) in the serum. RESULTS: After confirming the adequacy of an ELISA for measuring the serum level of sMET, we found no correlation between this level and the concentration of MET in tumors, as evaluated by immunohistochemistry and western blotting. Nevertheless, all but one tumor displaying a high MET level also displayed receptor phosphorylation, restricted to a small number of tumor cells. CONCLUSION: Our results thus demonstrate that the serum level of sMET is not indicative of the amount of MET present in the tumor cells and cannot be used as a biomarker for therapeutic purposes. However, MET scoring of tumor biopsies could be a first step prior to determination of MET receptor activation in high-MET tumors.

Milk growth factors and expression of small intestinal growth factor receptors during the perinatal period in mice.

BACKGROUND: Growth factors (GFs) are milk bioactive components contributing to the regulation of neonatal small intestinal maturation, and their receptors on the small intestinal epithelium play essential roles in mediating the functions of GFs. There is limited data correlating milk GFs and their receptors in the neonatal small intestine during the perinatal period. METHODS: Small intestines of C57BL/6N mouse pups were collected at regular intervals during fetal life and up to postnatal day (PD) 60. Gene expression of GF receptors was determined by real-time qPCR. Milk GF concentrations up to PD21 were analyzed by enzyme-linked immunosorbent assay. RESULTS: The majority of GF receptors showed significantly greater expression in the fetus than in postnatal life, and a sharp decrease occurred from PD14 extending to PD60; solid food restriction (PD14 and PD18) did not affect this decrease. Concentrations of five detected milk GFs demonstrated that GFs and the corresponding small intestinal receptors exhibited different correlations, with only milk transforming growth factor ß1 (TGF-ß1) having a significant positive correlation with TGF-ß receptor 1 mRNA. CONCLUSION: Gene expression of small intestinal GF receptors is likely a process of neonatal intestinal maturation that is affected concurrently by milk GFs and additional endogenous factors.

Tumor and Plasma Met Levels in Non-Metastatic Prostate Cancer.

OBJECTIVE: To measure Met protein content in prostate biopsies guided by fused magnetic resonance and ultrasound imaging, and to measure soluble Met (sMet) protein concentration in plasma samples from patients presenting evidence of prostate cancer. PATIENTS AND METHODS: 345 patients had plasma samples drawn prior to image-guided biopsy of the prostate. Of these, 32% had benign biopsies. Of the 236 that were positive for prostate adenocarcinoma (PCa), 132 treated by total prostatectomy had Gleason scores of 6 (17%), 7, (55%), 8 (16%), or 9-10 (12%). 23% had evidence of local invasion. Plasma samples were also obtained from 80 healthy volunteers. Tissue Met and plasma sMet were measured by two-site immunoassay; values were compared among clinically defined groups using non-parametric statistical tests to determine significant differences or correlations. RESULTS: PCa tumor Met correlated significantly with plasma sMet, but median values were similar among benign and malignant groups. Median plasma sMet values were also similar among those groups, although both medians were significantly above normal. Median Met content in primary PCa tumors and sMet concentrations were independent of Gleason score, final pathologic stage and age. CONCLUSION: Plasma sMet is not predictive of PCa or its severity in patients with organ-confined or locally invasive disease. Quantitative analysis of Met protein content and activation state in PCa tumor biopsy samples was highly feasible and may have value in follow-up to genomic and/or transcriptomic-based screens that show evidence of oncogenically relevant MET gene features that occur at relatively low frequency in non-metastatic PCa.

Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress.

Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.