Correlation of circulating tumor DNA EGFR mutation levels with clinical outcomes in patients with advanced lung adenocarcinoma.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for non-invasive epidermal growth factor receptor mutations (EGFRm) detection in lung cancer patients, but existing methods have limitations in sensitivity and availability. In this study, we used the ΔCt value (mutant cycle threshold [Ct] value-internal control Ct value) generated during the polymerase chain reaction (PCR) assay to convert super-amplification-refractory mutation system (superARMS) from a qualitative method to a semi-quantitative method named reformed-superARMS (R-superARMS), and evaluated its performance in detecting EGFRm in plasma ctDNA in patients with advanced lung adenocarcinoma. METHODS: A total of 41 pairs of tissues and plasma samples were obtained from lung adenocarcinoma patients who had known EGFRm in tumor tissue and were previously untreated. EGFRm in ctDNA was identified by using superARMS. Through making use of ΔCt value generated during the detection process of superARMS, we indirectly transform this qualitative detection method into a semi-quantitative PCR detection method, named R-superARMS. Both qualitative and quantitative analyses of the data were performed. Kaplan-Meier analysis was performed to estimate the progression-free survival (PFS) and overall survival (OS). Fisher exact test was used for categorical variables. RESULTS: The concordance rate of EGFRm in tumor tissues and matched plasma samples was 68.3% (28/41). At baseline, EGFRm-positive patients were divided into two groups according to the cut-off ΔCt value of EGFRm set at 8.11. A significant difference in the median OS (mOS) between the two groups was observed (EGFRm ΔCt ≤8.11 vs. >8.11: not reached vs. 11.0 months; log-rank P = 0.024). Patients were divided into mutation clearance (MC) group and mutation incomplete clearance (MIC) group according to whether the ΔCt value of EGFRm test turned negative after 1 month of treatment. We found that there was also a significant difference in mOS (not reached vs. 10.4 months; log-rank P = 0.021) between MC group and MIC group. Although there was no significant difference in PFS between the two groups, the two curves were separated and the PFS of MC group tended to be higher than the MIC group (not reached vs. 27.5 months; log-rank P = 0.088). Furthermore, EGFRm-positive patients were divided into two groups according to the cut-off of the changes in ΔCt value of EGFRm after 1 month of treatment, which was set at 4.89. A significant difference in the mOS between the two groups was observed (change value of ΔCt >4.89 vs. ≤4.89: not reached vs. 11.0 months; log-rank P = 0.014). CONCLUSIONS: Detecting EGFRm in ctDNA using R-superARMS can identify patients who are more likely sensitive to targeted therapy, reflect the molecular load of patients, and predict the therapeutic efficacy and clinical outcomes of patients.

Dysfunction in superior frontal gyrus associated with diphasic dyskinesia in Parkinson's disease.

Alterations in brain function in Parkinson's disease (PD) patients with diphasic dyskinesia have not been investigated. We aimed to explore the alterations in regional brain function. Each of 53 levodopa (LD)-treated PD patients had two resting-state functional magnetic resonance imaging (rs-fMRI) scans in the same morning, before and after taking LD. The regional homogeneity (ReHo) approach was used to reveal local synchronization changes. Two-way factorial repeated measures analysis of covariance, with group as a between-subject factor and LD effect as a within-subject factor, was performed to explore the two main effects and interaction. Interactive analysis was used to show outcomes that combined disease status and LD effect. Spearman's correlations were used to detect associations between interactive brain regions and severity of dyskinetic symptoms, assessed by the Unified Dyskinesia Rating Scale (UDyRS) scores, along with moderation analyses. There was no significant difference in the main group effect analysis. Significantly different clusters obtained from main LD effect analysis were in left caudate nucleus and putamen. ReHo values decreased in the caudate nucleus and increased in the putamen during the ON state after taking LD. Interaction between group and LD effect was found in left medial superior frontal gyrus (mSFG), where there were the lowest ReHo values, and was negatively correlated with UDyRS scores in the diphasic dyskinetic group during the ON state. The relationship was independent of LD dose. Abnormal local synchronization in the mSFG is closely associated with the development of diphasic dyskinesia in PD patients.

The increased gray matter volumes of precentral gyri in Parkinson's disease patients with diphasic dyskinesia.

Abnormal dopaminergic modulation of the cortico-basal ganglia motor loops results in the emergence of levodopa-induced dyskinesia (LID). We focused on alterations in the gray matter (GM) volume and the cortical thickness of the brain, especially in cortico-basal ganglia motor loops, in Parkinson's disease (PD) with diphasic dyskinesia. 48 PD patients with diphasic dyskinesia, 60 PD patients without dyskinesia and 48 healthy controls (HC) were included. Voxel-based morphometry (VBM) was applied to get GM images from MRI brain images. FreeSurfer was used to get cortical thickness. Distinct analyses of covariance (ANCOVA) and linear contrasts were performed for early- and late-onset PD groups. The severity of diphasic dyskinesia was evaluated by the Unified Dyskinesia Rating Scale (UDysRS). Finally, the correlations between mean volumes of clusters showing differences and the UDysRS scores were performed by Pearson's correlation. The GM volumes of precentral gyri were increased in PD patients with diphasic dyskinesia when compared with those without dyskinesia, which were positively correlated with UDysRS scores in PD patients with diphasic dyskinesia. However, there was no significant difference in cortical thickness among groups. The increased precentral gyri GM volumes might be associated with the pathogenesis and the severity of diphasic dyskinesia.

Isolation and characterization of adult mammary stem cells from breast cancer-adjacent tissues.

Normal adult mammary stem cells (AMSCs) are promising sources for breast reconstruction, particularly following the resection of breast tumors. However, carcinogenic events can potentially convert normal AMSCs to cancer stem cells, posing a safety concern for the use of AMSCs for clinical tissue regeneration. In the present study, AMSCs and autologous primary breast cancer cells were isolated and compared for their ability to differentiate, their gene expression profile, and their potential to form tumors in vivo. AMSCs were isolated from normal tissue surrounding primary breast tumors by immunomagnetic sorting. The pluripotency of these cells was investigated by differentiation analysis, and gene expression profiles were compared with microarrays. Differentially expressed candidate genes were confirmed by reverse transcription-polymerase chain reaction and western blot analyses. The in vivo tumorigenicity of these cells, compared with low-malignancy MCF-7 cells, was also investigated by xenograft tumor formation analysis. The results revealed that AMSCs isolated from normal tissues surrounding primary breast tumors were positive for the stem cell markers epithelial-specific antigen and keratin-19. When stimulated with basic fibroblast growth factor, a differentiation agent, these AMSCs formed lobuloalveolar structures with myoepithelia that were positive for common acute lymphoblastic leukemia antigen. The gene expression profiles revealed that, compared with cancer cells, AMSCs expressed low levels of oncogenes, including MYC, RAS and ErbB receptor tyrosine kinase 2, and high levels of tumor suppressor genes, including RB transcriptional corepressor 1, phosphatase and tensin homolog, and cyclin-dependent kinase inhibitor 2A. When injected into nude non-obese diabetic/severe combined immunodeficiency-type mice, the AMSCs did not form tumors, and regular mammary ductal structures were generated. The AMSCs isolated from normal tissue adjacent to primary breast tumors had the normal phenotype of mammary stem cells, and therefore may be promising candidates for mammary reconstruction subsequent to breast tumor resection.

Anterior mediastinum invasion by multiple myeloma: A case report.

Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells in the bone marrow (BM) that secretes monoclonal paraproteins in the blood serum and urine. Bone marrow MM cells can invade and damage the functions of other tissues and organs, such as the lungs, spleen, liver, pancreas, kidneys and lymph nodes. However, the invasion of MM cells primarily located in the BM to the anterior mediastinum at the site of the thymus is an extremely rare event. The current study reports the case of a 53-year-old female who presented with MM with involvement of the anterior mediastinum. The diagnosis was based on clinical imaging analyses and the results from BM and laboratory examinations, local biopsy pathology and immunohistochemistry. The patient was administered two courses of chemotherapy (epirubicin, dexamethasone and thalidomide). As a result, the tumor reduced in size, but the laboratory examination indicated no significant change. Next, the patient was switched to one course of PAD chemotherapy (bortezomib, epirubicin and dexamethasone). The original tumor was significantly reduced in size following this chemotherapy, and all the indicators improved. The present study suggests that invasion of the thymus by MM may lead to immune disturbance arising from the abnormal thymus gland. In the clinic, extramedullary plasmacytoma in the thymus should be carefully distinguished from thymoma.

Clinicopathological characteristics and prognosis of patients with adenosquamous lung carcinoma.

This study was aimed to characterize clinicopathological features and prognosis of patients with adenosquamous lung carcinoma (ASC). Among the 2531 patients with lung cancer who underwent surgery between January 2000 and June 2012 in our hospital, 59 were histologically diagnosed as having ASC. The clinicopathological features and follow-up data of ASC patients were collected and analyzed statistically. Superior lobectomy was accomplished in 40 patients, middle and inferior lobectomy in 3, lobectomy plus partial resection of contralateral lung in 5, partial lung resection in 4, and pneumonectomy in 7. Moreover, 22 cases were found to be adenocarcinoma-predominant, and 18 to be squamous cell carcinoma-predominant. The median survival time was 13.6 months, and the 1-, 3-, and 5-year survival rates were 59.9%, 36.4% and 31.2%, respectively. Of the 52 cases with tissue specimens available, 11 had an EGFR mutation (21.2%) and 2 had a KRAS mutation (3.8%). Multivariate analysis showed that histology subtype, pleural invasion, TNM stage, and postoperative treatment were all independent prognostic factors. The data from the current study demonstrated that SCC-predominant histology represents a better prognosis of ASC. Histology subtype, pleural invasion, TNM stage, and postoperative treatment are independent prognostic factors for ASC and adjuvant therapy may help control the disease.

MDM2 rs2279744 and TP53 rs1042522 polymorphisms associated with etoposide- and cisplatin-induced grade III/IV neutropenia in Chinese extensive-stage small-cell lung cancer patients.

BACKGROUND/AIMS: Etoposide and cisplatin (EP) chemotherapy is the most frequently used regimen in extensive-stage small-cell lung cancer (SCLC) patients, although the side effects (e.g., neutropenia) are high. This study investigates the association of the MDM2 rs2279744 and TP53 rs1042522 single-nucleotide polymorphisms (SNPs) with EP-induced grade III/IV neutropenia and with response to EP in extensive-stage SCLC patients. METHODS: Blood samples from 119 extensive-stage SCLC patients were subjected to genotyping of these 2 SNPs, using the allele-specific matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry for determining the association with neutropenia in the patients. RESULTS: The data showed that patients carrying the MDM2 rs2279744-GG genotype were associated with a lower incidence of grade III/IV neutropenia in the recessive and additive models, while the TP53 rs1042522-CC genotype was associated with a higher incidence in the recessive model. Furthermore, the combination of the MDM2 rs2279744-TT+TG and the TP53 rs1042522-CC genotype was associated with a significantly higher incidence of grade III/IV neutropenia. And the combination of the MDM2 rs2279744-GG and the TP53 rs1042522-GG+GC genotype was associated with the lowest incidence of grade III/IV neutropenia. CONCLUSIONS: MDM2 rs2279744 and TP53 rs1042522 SNPs were associated with EP-induced high-grade neutropenia in extensive-stage SCLC patients. Further studies are needed to investigate the underlying mechanisms.

[Study on the mechanism of arsenic trioxide inhibiting NB4 cells proliferation].

OBJECTIVE: To explore the molecular mechanisms of arsenic trioxide (As2O3) inhibiting NB4 cells proliferation. METHODS: The Janus kinase 1 (JAK1) protein level and its phosphorylation level in NB4 cells was detected by Western blots. NB4 cells were transfected with JAK1 siRNA or JAK1 plasmid to make JAK1 gene silenced or overexpressed. The inhibition of NB4 cells proliferation was measured by MTT assay and Trypan blue exclusion respectively. The variation of phosphorylation level of JAK1 and the cell cycle inhibitor P21 were determined by Western blots. RESULTS: JAK1 protein was expressed stably in NB4 cells, with no phosphorylation. The phosphorylation of JAK1 was enhanced after the NB4 cells treated with As2O3. After NB4 cells transfected with JAK1 siRNA, the expression level of JAK1 was obviously lower than that of in the non-specific siRNA group and blank control group. The effect of As2O3 inhibiting NB4 cells proliferation was weaker in the JAK1 siRNA transfected group. The inhibiting rate of 4 micromol/L As2O3 on NB4 cells proliferation of JAK1 siRNA group was 49.12% being lower than that of the non-specific siRNA group (74.58%) and control group (72.33%). After NB4 cells transfected with JAK1 plasmid, the JAK1 expression level in wild-type and mutant type plasmid groups were significantly higher than those in the empty plasmid group, moreover the effect of As2O3 inhibiting proliferation was stronger in wild-type plasmid group. The inhibiting rate of 4 micromol/L As2O3 on NB4 cells proliferation of wild-type plasmid group was 69.53% being higher than that of the mutant type JAK1 plasmid group (37.26%) and the empty plasmid group (39.61%). The expression level of P21 was up-regulated after the NB4 cells treated with As2O3. CONCLUSION: JAK1 is expressed stably in NB4 cells, but has no activity. Arsenic trioxide inhibits the proliferation of NB4 cells through activating the JAK1. P21 is up-regulated after arsenic trioxide activated the JAK1 to inhibit the proliferation of NB4 cells.

[Mechanism of leukemia cell apoptosis induced by sodium butyrate activating TRAIL pathway].

This study was aimed to investigate the mechanism of leukemia cell apoptosis induced by histone deacetylase inhibitor (HDACI). Flow cytometry was used to detect the apoptosis of leukemia cell lines NB4, U937 and Jurkat, and the changes of mRNA and protein expressions of TRAIL, DR4 and DR5 were detected by Western blot and RT-PCR respectively. The results showed that both TRAIL and DR5 protein and mRNA expressions in NB4, U937 and Jurkat cells increased after treated with sodium butyrate (SB) and in time-dependent manner. However, DR4 mRNA in leukemia cells was not significantly changed after treated with SB. It is concluded that the apoptosis mechanism of leukemic cell lines NB4, U937 and Jurkat induced by SB is closely related to the protein and mRNA expressions up-regulating TRAIL and DR5, but the DR4 may not participate in the apoptosis induced by SB.