HOXA9 gene promotor methylation and copy number variation of SOX2 and HV2 genes in cell free DNA: A potential diagnostic panel for non-small cell lung cancer.

BACKGROUND: Most cases of lung cancer are diagnosed at advanced stage. Detection of genetic and epigenetic markers in cell-free DNA (cfDNA) is a promising tool for the diagnosis of lung cancer at an early stage. The aim of this study was to identify non-invasive diagnostic markers in cell free DNA (cfDNA) for non-small cell lung cancer (NSCLC) as it is the most common type of lung cancer. METHODS: We investigated the cfDNA HOXA9 gene promotor methylation by pyrosequencing. Copy number variation of SOX2 and HV2 genes were detected by real-time PCR in cfDNA extracted from plasma samples of 25 newly diagnosed NSCLC patients and 25 age and sex matched controls. RESULTS: Methylation level of HOXA9 was significantly higher in NSCLC patients than controls (p > 0.001). SOX2 showed significantly higher CNV and HV2 showed lower CNV in patients than controls (p > 0.001, p = 0.001 respectively). Receiver Operating Characteristic (ROC) curve analysis for HOXA9 methylation, SOX2 CNV and HV2 CNV showed a discrimination power of 79.4%, 80% and 77.5% respectively and the area under the curve for the combined analysis of the three genes was 0.958 with 88% sensitivity and 100% specificity. CONCLUSIONS: In this study, we suggest a potentially diagnostic panel that may help in detection of lung cancer with high sensitivity and specificity using cell free DNA. This Panel included HOXA9 gene methylation and the CNV of SOX2 and HV2 genes.

Serum proteome profiling reveals SOX3 as a candidate prognostic marker for gastric cancer.

Searching for the novel tumour biomarkers is pressing for gastric cancer diagnostication and prognostication. The serum specimens from patients diagnosed with locally advanced gastric carcinoma before operation and 4 week after surgery were collected, respectively, and serum proteome profiling was conducted by liquid chromatography-mass spectrometry (MS)/MS. Fifty-five proteins were identified to be up-regulated and 16 proteins were down-regulated, and these differentially expressed proteins participated in various biological processes. Serum levels of SOX3, one of down-regulated proteins, in stomach cancer patients were higher than in healthy controls. SOX3 levels in cancer tissues were remarkably related to tumour differentiation, lymph node metastasis, primary tumour invasion and pTNM (pathological TNM) stage. Analysis with The Cancer Genome Atlas database indicated that SOX3 level and pTNM stage were the independent risk factors for the patient survival and that the overall survival was negatively associated with the SOX3 levels. Loss-of-function showed that SOX3 promoted gastric cancer cell invasion and migration in vitro and in vivo. SOX3 silence inhibits the expression of MMP9, and SOX3 is responsible for MMP9 expression transcriptionally. Our study highlights the potentiality of the paired pre- and post-operation serum proteome signatures for the detection of biomarkers and reveals that SOX3 may serve as a candidate prognosis marker for gastric cancer.

A rare case of long-term paraesthesia diagnosed as a paraneoplastic syndrome by anti-SOX1 antibody determination.

Paraneoplastic syndromes (PS) are a rare presentation of cancer, most commonly associated with small cell lung cancer (SCLC), breast cancer and haematologic malignancies. The diagnosis of PS is challenging because it could affect multiple organ systems and it may present before the tumour is visible by imaging. We report a malignant tumour diagnosed in a male patient who referred long-term paraesthesia and proximal muscle strength loss. After ruling out common causes of polyneuropathy, the anti-SOX1 antibody gave light to the diagnosis. A pulmonary opacity in the upper right lobe was observed in the chest X-ray and a pulmonary tumour was later confirmed by CT scan. The biopsy of the cervical lymphadenopathy determined an SCLC, which caused a PS called Lambert-Eaton myasthenic syndrome (LEMS). Our case raises awareness of a rare PS presentation, which can be diagnosed by specific antibodies, allowing early diagnosis and treatment of lung cancer.

Highly sensitive and cost-effective ITO-based immunosensor system modified by 11-CUTMS: Analysis of SOX2 protein in real human serum.

In this study it is aimed to construct an immunosensor system for detection of Sex-determining region Y-box 2 (SOX2) antigen by using Indium tin oxide- polyethylene terephthalate (ITO-PET) as working electrode. Firstly, ITO-PET electrode surfaces were hydroxylated by using NH4OH/H2O2/H2O and were incubated with 11-cyanoundecyltrimethoxysilane (11-CUTMS), respectively. After silanization process, anti- SOX2 was immobilized on modified ITO-PET electrodes. All immobilization processes were examined with Electrochemical impedance spectroscopy (EIS) and Cyclic voltammetry (CV). The basic parameters such as 11-CUTMS and anti-SOX2 concentrations, anti-SOX2 and SOX2 incubation period were optimized. The immunosensor prepared under optimal conditions gave a response for a wide concentration range of SOX2 protein (0.02 pg mL-1-2 pg mL-1) and the limit of detection was determined as low as 0.013 pg mL-1. And also, the immunosensor had good repeatability, reproducibility, reusability and long shelf life. Scanning Electron Microscope (SEM) method was utilized to observe the morphologies of the electrode surfaces belonging to all steps. Lastly, seven different real human serum were analyzed with the constructed immunosensor and Enzyme-Linked ImmunoSorbent Assay (ELISA). The results found with these methods were analogised with each other.

Complex Analysis of Total and Fetal DNA and Cytokines in Blood Plasma of Pregnant Women with Preeclampsia.

We performed a complex analysis of total and fetal extracellular DNA, 8 cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage CSF, IFNγ, and TNFα) in blood plasma obtained from women with preeclampsia prior to labor onset. Total (sensitivity 89.47%, specificity 93.75%) and fetal extracellular DNA (sensitivity 73.68%, specificity 87.5%) were the most accurate parameters determining preeclampsia. We revealed a high correlation (p=3×10-6) between total and fetal extracellular DNA levels in the group of preeclampsia. Preeclampsia significantly increased the levels of macrophage factors IL-10 and IL-6. These cytokines significantly correlated with the levels of total and fetal extracellular DNA in the preeclampsia group. In the control group, such correlations were not observed. These findings obtained suggest that preeclampsia develops upon increased macrophage activity, leading to destruction of the placenta trophoblast cells.

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women.

Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46- 2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10-4-10-3 ng/µL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

[The expression and significance of stem cell transcription factor Sox2 in lung carcinoma].

BACKGROUND AND OBJECTIVE: Transcription factor Sox2 remains the pluripotency of stem cell, participates in self-renew of cancer stem cell and plays important role during the initiation and development of various cancers. This study intends to investigate the expression and significance of Sox2 and Sox2 autoantibodies (Sox2-Ab) in tissue and serum of patients with non-small cell lung cancer (NSCLC). METHODS: Expression of Sox2 gene and protein was tested in 58 cases of NSCLC, 16 patients with other tumors and 20 cases of normal lung tissue specimens by quantitative PCR and immunohistochemical assay, respectively. Serum Sox2-Ab level was detected in 30 cases of NSCLC patients and 30 healthy controls by ELISA method. Clinical and pathological data from patients were collected and analyzed retrospectively. RESULTS: Expression levels of Sox2 mRNA and protein were higher in patient with NSCLC than other groups, with statistically significant differences (P<0.01), respectively. Meanwhile, Sox2 mRNA expression increased in NSCLC patients associated with histological type and tumor size. No significant differences in Sox2-Ab serum levels were found between NSCLC patients and normal subjects. CONCLUSIONS: Sox2 in NSCLC have a higher expression, which is closely related to histological type and tumor size. Sox2 might use as a potential biomarker and therapeutic target for lung cancer.

Prognostic value of changes in the expression of stem cell markers in the peripheral blood of patients with colon cancer.

Cancer stem cells play an important role in carcinogenesis and resistance to treatment and may lead to metastasis. The isolation of circulating stem cells involves cell sorting based on the presence of cell surface markers. Many surface markers such as CD133, c-Kit, SOX, OCT4 and TWIST have been reported. In the present study, we determined the expression of different stem cell markers and their variation in expression at different stages of the treatment process. Samples of EDTA blood were collected from metastatic colorectal cancer patients, and circulating cancer stem cells were isolated for the analysis of the expression of stem cell markers using RT-PCR. These findings were correlated with the response to therapy. All statistical analyses were performed using the GraphPad Prism 5.03 software. Significant differences were found in the expression levels of the markers CD133, SOX2, OCT4 and TWIST1. No differences were found in c-Kit expression. Correlation in the expression levels of most of the markers was observed. Expression of CD133, OCT4, SOX2 and TWIST1 had a predictive value for colon cancer behavior. Evaluation of this stem cell gene expression panel may be useful for predicting the response during the process of treatment, and the relative easy access to samples facilitates this method. Moreover the correlation between CD133 and TWIST1 expression may be associated with tumor regrowth and metastatic relapse.

Identification of novel SRY mutations and SF1 (NR5A1) changes in patients with pure gonadal dysgenesis and 46,XY karyotype.

Primary amenorrhea due to 46,XY disorders of sexual development (DSD) is complex with the involvement of several genes. Karyotyping of such patients is important as they may develop dysgerminoma and molecular analysis is important to identify the underlying mechanism and explore the cascade of events occurring during sexual development. The present study was undertaken for the genetic analysis in seven patients from five families presenting with primary amenorrhea and diagnosed with pure gonadal dysgenesis. Karyotyping was done and the patients were screened for underlying changes in SRY, desert hedgehog (DHH), DAX1 (NR0B1) and SF1 (NR5A1) genes, mutations in which are implicated in DSD. All the patients had 46,XY karyotype and two novel SRY mutations were found. In Family 1 (Patient S1.1) a missense mutation c.294G>A was seen, which results in a stop codon at the corresponding amino acid (Trp98X) and in Family 2 (Patients S2.1, S2.2 and S2.3), a missense mutation c.334G>A (Glu112Leu) was identified in all affected sisters. Both mutations were seen to occur in the conserved high mobility group box of SRY gene. One heterozygous change c.427G>A resulting in Glu143Lys in DHH gene in one patient and two heterozygous changes in the intronic region of SF1 (NR5A1) gene (c.244+80G>A+ c.1068-20C>T) in another patient were noted. One individual did not show changes in any of the genes analyzed. These results reiterate the importance of SRY and others, such as SF1 (NR5A1) and DHH, that are involved in the cascade of events leading to sex determination and also their role in sex reversal.