Longitudinal Plasma Proteomics-Derived Biomarkers Predict Response to MET Inhibitors for MET-Dysregulated NSCLC.

MET inhibitors have shown promising efficacy for MET-dysregulated non-small cell lung cancer (NSCLC). However, quite a few patients cannot benefit from it due to the lack of powerful biomarkers. This study aims to explore the potential role of plasma proteomics-derived biomarkers for patients treated with MET inhibitors using mass spectrometry. We analyzed the plasma proteomics from patients with MET dysregulation (including MET amplification and MET overexpression) treated with MET inhibitors. Thirty-three MET-dysregulated NSCLC patients with longitudinal 89 plasma samples were included. We classified patients into the PD group and non-PD group based on clinical response. The baseline proteomic profiles of patients in the PD group were distinct from those in the non-PD group. Through protein screening, we found that a four-protein signature (MYH9, GNB1, ALOX12B, HSD17B4) could predict the efficacy of patients treated with MET inhibitors, with an area under the curve (AUC) of 0.93, better than conventional fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC) tests. In addition, combining the four-protein signature with FISH or IHC test could also reach higher predictive performance. Further, the combined signature could predict progression-free survival for MET-dysregulated NSCLC (p < 0.001). We also validated the performance of the four-protein signature in another cohort of plasma using an enzyme-linked immunosorbent assay. In conclusion, the four plasma protein signature (MYH9, GNB1, ALOX12B, and HSD17B4 proteins) might play a substitutable or complementary role to conventional MET FISH or IHC tests. This exploration will help select patients who may benefit from MET inhibitors.

Prognostic features and comprehensive genomic analysis of NF1 mutations in EGFR mutant lung cancer patients.

OBJECTIVE: NF1 is a tumor suppressor gene that encodes the neurofibromin protein and negatively regulates Ras signaling. This study was aimed to investigate the molecular, clinical characteristics, and prognostic features of NF1 gene in EGFR mutant lung cancer patients. METHOD: The next-generation sequencing (NGS) was used to analyze the data from lung cancer patients in the Guangdong Lung Cancer Institute (GLCI) from June 2016 to December 2020. RESULTS: Somatic NF1 mutations were present in 4.2% (135/3220) of Chinese lung cancer patients. NF1 mutations where clearly enriched in older (p < 0.001), male (p < 0.001), and smoking (p < 0.001) patients. Patients with NF1 mutations were more likely to have TP53 (p = 0.003), BRAF (p = 0.001) and RASA1 (p = 0.026) mutations and mutually exclusive with EGFR mutations (p = 0.006). TP53 mutation had worsen prognosis in cases of NF1 mutant (p = 0.026) or EGFR/NF1 co-mutant (p = 0.031) lung adenocarcinomas (LUAD) patients. There was no effect on overall survival (OS) in LUAD patients with and without NF1 mutations, even in LUAD driver-gene negative patients. NF1/EGFR co-mutation patients had a longer OS than a single mutation of either the EGFR gene (median OS: 47.7 m vs. 30.2 m, hazard ratio [95% CI], 0.47 [0.30-0.74], p = 0.004) or NF1 gene (47.7 m vs. 19.0 m, 0.44 [0.27-0.73], p = 0.003). Furthermore, NF1 mutations significantly prolonged OS in EGFR mutant/TP53 wild-type LUAD patients (106.5 m vs. 25.5 m, 0.28 [0.13-0.59], p = 0.003) but not in patients with EGFR/TP53 co-mutations (36.8 m vs. 30.2 m, 0.70 [0.39-1.26], p = 0.280). CONCLUSION: Our results indicated NF1 mutations served as a good prognostic factor in EGFR mutant/TP53 wild-type lung cancer patients in this single-center study. TP53 mutation was obviously enriched in NF1 mutant patients and had shorter OS.

Pregnancy and fetal outcomes of chronic hepatitis C mothers with viremia in China.

BACKGROUND: Data that assess maternal and infant outcomes in hepatitis C virus (HCV)-infected mothers are limited. AIM: To investigate the frequency of complications and the associated risk factors. METHODS: We performed a cohort study to compare pregnancy and fetal outcomes of HCV-viremic mothers with those of healthy mothers. Risk factors were analyzed with logistic regression. RESULTS: Among 112 consecutive HCV antibody-positive mothers screened, we enrolled 79 viremic mothers. We randomly selected 115 healthy mothers from the birth registry as the control. Compared to healthy mothers, HCV mothers had a significantly higher frequency of anemia [2.6% (3/115) vs 19.0% (15/79), P < 0.001] during pregnancy, medical conditions that required caesarian section [27.8% (32/115) vs 48.1% (38/79), P = 0.004], and nuchal cords [9.6% (11/115) vs 34.2% (27/79), P < 0.001]. In addition, the mean neonatal weight in the HCV group was significantly lower (3278.3 ± 462.0 vs 3105.1 ± 459.4 gms; P = 0.006), and the mean head circumference was smaller (33.3 ± 0.6 vs 33.1 ± 0.7 cm; P = 0.03). In a multivariate model, HCV-infected mothers were more likely to suffer anemia [adjusted odds ratio (OR): 18.1, 95% confidence interval (CI): 4.3-76.6], require caesarian sections (adjusted OR: 2.6, 95%CI: 1.4-4.9), and have nuchal cords (adjusted OR: 5.6, 95%CI: 2.4-13.0). Their neonates were also more likely to have smaller head circumferences (adjusted OR: 2.1, 95%CI: 1.1-4.3) and lower birth weights than the average (≤ 3250 gms) with an adjusted OR of 2.2 (95%CI: 1.2-4.0). The vertical transmission rate was 1% in HCV-infected mothers. CONCLUSION: Maternal HCV infections may associate with pregnancy and obstetric complications. We demonstrated a previously unreported association between maternal HCV viremia and a smaller neonatal head circumference, suggesting fetal growth restriction.

Clinical characteristics and sequence complexity of anaplastic lymphoma kinase gene fusions in Chinese lung cancer patients.

OBJECTIVES: To investigate the clinical characteristics of anaplastic lymphoma kinase (ALK) rearrangements and sequence complexity of the ALK fusion gene in Chinese lung cancer patients. METHODS: We prospectively screened ALK rearrangements in 1474 lung cancer specimens, including 1387 cases of non-small cell lung cancer (NSCLC), 54 cases of small cell lung cancer (SCLC), and 33 cases of cancer with lung metastasis from other organs by both standard polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE)-coupled PCR. Fifteen cases of ALK-positive RACE-coupled PCR products were transformed into Escherichia coli for molecular cloning and sequenced for complexity analysis. RESULTS: The overall frequency of ALK rearrangements was 5.1% (71/1387) in NSCLC. In 71 positive cases, the coexistence of epidermal growth factor receptor (EGFR) and ALK variations was found in 6 cases (8.5%), and the coexistence of different ALK variants was found in 2 cases (2.8%) (1 case with variants 1 and 9; the other case with variants 3 and 2) by PCR analysis. Furthermore, through sequence cloning analysis of 15 cases of non-selective ALK-positive samples, two cases with variants 1 and 3 harbored the coexistence of three subtypes (variant 1 subtypes: E13; A20, E13del63; A20 and E7E12E13; A20 and variant 3 subtypes: E6; A20, E6ins33; A20 and E3E6; A20). Variant 3a and 3b subtypes were always coexistent and had the same proportion of ALK variant 3 rearrangements. ALK rearrangement was associated with young age, female gender, never-smokers, those with adenocarcinoma, advanced stage, and EGFR mutations. No ALK fusion was detected in 54 cases of SCLC or 33 cases of cancer with lung metastasis from other organs. CONCLUSIONS: The identification of novel ALK variants, the coexistence of EGFR mutations and ALK fusions, the coexistence of ALK variants, and the coexistence of subtypes reveal the diversity and sequence complexity of ALK fusions.

Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

BACKGROUND: Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform. METHODS: A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility. RESULTS: Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform. CONCLUSIONS: We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.

The critical roles of mitophagy in cerebral ischemia.

Mitochondria play a key role in various cell processes including ATP production, Ca2+ homeostasis, reactive oxygen species (ROS) generation, and apoptosis. The selective removal of impaired mitochondria by autophagosome is known as mitophagy. Cerebral ischemia is a common form of stroke caused by insufficient blood supply to the brain. Emerging evidence suggests that mitophagy plays important roles in the pathophysiological process of cerebral ischemia. This review focuses on the relationship between ischemic brain injury and mitophagy. Based on the latest research, it describes how the signaling pathways of mitophagy appear to be involved in cerebral ischemia.

Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform.

OBJECTIVE: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on MassARRAY mass spectrometry platform. METHODS: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of 99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a LungCarta(TM) kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. RESULTS: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with LungCarta(TM) kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively. CONCLUSIONS: The proposed MassARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues.

Anaplastic Lymphoma Kinase Variants and the Percentage of ALK-Positive Tumor Cells and the Efficacy of Crizotinib in Advanced NSCLC.

BACKGROUND: Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer patients exhibited heterogeneous magnitude of response and duration time to criztotinib treatment. This study explored ALK variants and the percentage of ALK-positive cells using fluorescent in situ hybridization (FISH) on clinical efficacy of crizotinib. PATIENTS AND METHODS: A total of 120 patients with ALK rearrangement who were treated with criztotinib were enrolled. ALK variants were clarified in 61 patients, and ALK percentages were evaluated using FISH in 114 ALK-positive patients. Retrospectively, objective response rate, and progression-free survival (PFS) were evaluated. RESULTS: A total of 61 patients with specific ALK variants were divided into 3 subgroups, echinoderm microtubule-associated protein like 4 (EML4)-ALK variant 1 (n = 22), EML4-ALK variant 3a/b (n = 18), and other ALK variants (n = 21). Median PFS in the 3 subgroups was 11.0 months (95% confidence interval [CI], 5.5-16.5), 10.9 months (95% CI, 5.9-15.8), 7.4 months (95% CI, 3.2-11.6), respectively, and no significant difference (P = .795) existed among them. The percentage of ALK-positive cells in FISH analysis was weakly correlated with PFS (rs = 0.235; P = .015). Additionally, it was also weakly correlated with best response to crizotinib (rs = 0.288; P = .003). Overall, there were 45, 49, and 26 patients receiving first, second, and third or further-line crizotinib, respectively. Median PFS in the first-line setting (10.5 months; 95% CI, 8.6-12.4) was significantly longer than that in the second-line setting (8.3 months; 95% CI, 4.7-12.0; P = .020). CONCLUSION: Anaplastic lymphoma kinase variants might have no correlation with clinical response to crizotinib. The percentage of ALK-positive cells might correlate with the extent of benefit from crizotinib treatment.

Lung cancers with concomitant EGFR mutations and ALK rearrangements: diverse responses to EGFR-TKI and crizotinib in relation to diverse receptors phosphorylation.

PURPOSE: We investigated the incidence of concomitant epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements in Chinese patients with non-small cell lung cancer (NSCLC), and assessed responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and crizotinib in such tumors. EXPERIMENTAL DESIGN: We screened 977 consecutive patients with NSCLC for the presence of concomitant EGFR mutations and ALK rearrangements by rapid amplification of cDNA ends-coupled PCR sequencing and FISH. Immunohistochemistry (IHC) and Western blotting were used to correlate the activation of EGFR, ALK, and downstream proteins with responses to EGFR-TKIs and crizotinib. RESULTS: The overall frequency of concomitant EGFR mutations and ALK rearrangements was 1.3% (13/977). EGFR/ALK co-alterations were found in 3.9% (13/336) EGFR-mutant and 18.6% (13/70) ALK-rearranged patients. Ten tumors were treated with first-line EGFR-TKIs, with a response rate of 80% (8/10). Two tumors with high phospho-ALK levels and low phospho-EGFR levels achieved stable and progressive disease, respectively. Median progression-free survival was 11.2 months. Coexpression of mutant EGFR and ALK fusion proteins in the same tumor cell populations was detected by IHC. Two cases with high phospho-ALK levels treated with crizotinib achieved partial responses; two cases with low phospho-ALK levels had progressive or stable disease. CONCLUSION: ALK rearrangements and EGFR mutations could coexist in a small subgroup of NSCLC. Advanced pulmonary adenocarcinomas with such co-alterations could have diverse responses to EGFR-TKIs and crizotinib. Relative phospho-ALK and phospho-EGFR levels could predict the efficacy of EGFR-TKI and crizotinib.

The biophysical property of A549 cells transferred by VEGF-D.

Vascular endothelial growth factor-D (VEGF-D) together with VEGF-C is considered to be associated with lymphangiogenesis and angiogenesis and involve in tumorization. This study aims to investigate the influence of exogenous VEGF-D gene on the biophysical property of cell surface of lung adenocarcinoma cell line. A panel of lung adenocarcinoma cell lines were examined the expression of VEGF-D and VEGF-C by real-time PCR. The VEGF-D recombinant plasmid containing enhanced green fluorescence protein (EGFP) was constructed and transfected to the cell line with no expression of VEGF-D and confirmed by real-time PCR and Western blot analysis. Topographic images of cells were obtained by using atomic force microscope (AFM) in contact mode. Unlike VEGF-C, VEGF-D was found to have a very low expression or undetectable expression in lung adenocarcinoma cell lines. The VEGF-D recombinant plasmid had been constructed successfully and was transferred into the human lung adenocarcinoma cell line A549 cells which had no endogenous expression of VEGF-D, and exogenous VEGF-D could be detected in mRNA and protein expression levels in the gene modified cells, while the VEGF-C gene expression had no change after VEGF-D transfection. After transfection, the irregular microspikes or nano clusters could observe on the surface of A549 cells, and VEGF-D transfected A549 cells became more rigid. The exogenous VEGF-D gene might cause the remarkable biophysical architectural changes in the A549 cells, which might as a novel biomarker for evaluation of its biological function.

[Effect of superantigen SEA on mitochondrial membrane potential of Molt-4 cell].

AIM: To explore the effect of superantigen staphylococcal enterotoxin A(SEA) on mitochondrial membrane potential of Molt-4 cell. METHODS: Cell counting kit-8(CCK-8) was used to detect the proliferation of T cell in different concentration and time, We employed JC-1 to estimate mitochondrialmembrane potential (δψm) of Molt-4 cell induced by SEA using flow cytometry (FCM). RESULTS: The proliferative activity of T cell treated with SEA(1 mg/L) was changed obviously compared with control.The mitochondrial membrane potential of Molt-4 cell with SEA(1 mg/L) was highest at 10 min by FCM after stimulation of 180, 60, 30 and 10 min. Mitochondrial membrane potential of Molt-4 cell treated with SEA after 10 and 30 min were lower than that with PHA which also have rising effect. CONCLUSION: Superantigen can enhance the mitochondrial membrane potential of Molt-4 cell in the early stage of proliferation.But the effect was weaker than that with mitogen PHA.

[Effect of superantigen SEA on the CD3epsilon chain expression on cord blood mononuclear cells stimulated by K562 cells].

AIM: To explore the effect of staphylococcal enterotoxin A(SEA) on CD3(epsilon) chain expression on mononuclear cells in cord blood, which were stimulated by K562 cells. METHODS: The anti-CD3 antibodies(mAb), K562 cells, SEA or both SEA and K562 cells were cocultured with mononuclear cells (MNCs) from four normal human cord blood for 48 hours respectively, Real-time PCR with SYBR Green I technique was used to detect expressed level of CD(epsilon) on MNCs in cord blood and set up a blank control group. Relative changes in expression level of CD3(epsilon) chain were indicated by the 2(-deltadeltaCt); method between each group and the control group. beta2-microglobulin gene(beta2M) was used as an endogenous reference. RESULTS: The expressed level of CD3(epsilon) chain on MNCs was slightly decreased in K562 cell group and were increased in the anti-CD3 antibodies mAb group, SEA group, both of SEA and K562 cell group respectively. The expressed level of CD3(epsilon) chain in both SEA and K562 group was significantly higher than that in the SEA group(P<0.01). CONCLUSION: Superantigen SEA can enhance the expressed level of CD3(epsilon) chain on MNCs stimulated by K562 cells.