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.

[Diagnostic Efficacy of SHOX2 Gene Hypermethylation for Lung Cancer: A Meta-Analysis].

BACKGROUND: Lung cancer is the most common malignant tumor in clinic. The prognosis of advanced patients is poor, and the 5-year survival rate is low. Therefore, early diagnosis becomes the key to improve the prognosis of patients. In recent years, with the development of molecular biology technology, aberrant modification of some driver genes, such as methylation, has become an important method for early diagnosis of lung cancer. The purpose of the present work was to quantitatively evaluate the diagnostic value of abnormal hypermethylation in short state homeobox 2 (SHOX2) promoter region in lung cancer by evidence-based medicine. METHODS: We searched MEDLINE, EMBASE, Ovid, Web of Science and CNKI for literatures related to the relationship between SHOX2 gene promoter hypermethylation and lung cancer. The data of SHOX2 promoter hymethylation in the original study were extracted. The diagnostic sensitivity, specificity and area under receiver operating characteristic (ROC) curve of SHOX2 promoter methylation were calculated. RESULTS: Finally, 13 publications were included in this meta-analysis, and due to significant statistical heterogeneity among the studies (P<0.05) the data was pooled by random effect model. The diagnostic sensitivity and specificity of SHOX2 promoter hypermethylation in the diagnosis of lung cancer were 0.75 (95%CI: 0.74-0.77) and 0.89 (95%CI: 0.88-0.91), respectively; The positive likelihood ratio value was 6.75 (4.56-9.99), and the negative predictive value was 0.36 (0.25-0.52); The diagnostic odds ratio was 23.16 (11.34-47.31), and the area under the ROC curve was 0.9. CONCLUSIONS: SHOX2 gene promoter hypermethylation is high in serum, broncholavage fluid and pleural effusion of lung cancer patients, which can be used as a biomarker for auxiliary diagnosis of lung cancer.

Plasma proteomic screening and validation of novel biomarkers in Takayasu's arteritis.

OBJECTIVES: To screen and validate differential proteins as novel biomarkers in active Takayasu's arteritis (TAK). METHODS: Plasma samples from 40 active, 40 inactive patients, and 40 healthy controls were collected. Protein profiles of plasma were mapped by two-dimensional gel electrophoresis. Differential protein spots were detected and identified by image analysis and mass spectrometry. Plasma concentrations of proteins were measured to validate candidate biomarkers. The area under the receiver operating characteristic (ROC) curve (AUC) of circulating plasma concentrations of candidate biomarkers were calculated to assess diagnostic value. RESULTS: With a total of 1507 matched gel spots, there were 170 differential expression spots between active and inactive TAK, including 139 up-regulated and 31 downregulated. Only 11 proteins could be identified by mass spectrometry. Serum amyloid A(SAA), fibrinogen, complement C4a, complement C3c, complement C4b binding protein(C4bp), recombination acting gene protein 1(RAG1), alpha-1-acid glycoprotein, alpha-1-microglobulin, complement C7, complement factor H related protein-1 were up-regulated in active patients, while serum amyloid P was down-regulated. Active patients had higher circulating levels of RAG1(P<0.001), C4bp (p=0.012) and SAA (p<0.001), compared to inactive patients, while inactive patients had higher levels than controls (RAG1, p=0.011; C4bp, p=0.012; SAA, p=0.005). The composite AUC with SAA, RAG1, and C4bp was 0.94 (95%CI 0.86-0.98) for discriminating activity, larger than 0.71(95% CI 0.60-0.80) for ESR (p=0.0004) or 0.75(95%CI 0.64-0.84) for CRP (p=0.0014), respectively. ONCLUSIONS: Some acute-phase and immunology-related proteins may serve as novel biomarkers of TAK. Further study of these proteins may be helpful to elucidate the pathologic mechanism.

A prediction model based on DNA methylation biomarkers and radiological characteristics for identifying malignant from benign pulmonary nodules.

BACKGROUND: Lung cancer remains the leading cause of cancer deaths across the world. Early detection of lung cancer by low-dose computed tomography (LDCT) can reduce the mortality rate. However, making a definitive preoperative diagnosis of malignant pulmonary nodules (PNs) found by LDCT is a clinical challenge. This study aimed to develop a prediction model based on DNA methylation biomarkers and radiological characteristics for identifying malignant pulmonary nodules from benign PNs. METHODS: We assessed three DNA methylation biomarkers (PTGER4, RASSF1A, and SHOX2) and clinically-relevant variables in a training cohort of 110 individuals with PNs. Four machine-learning-based prediction models were established and compared, including the K-nearest neighbors (KNN), random forest (RF), support vector machine (SVM), and logistic regression (LR) algorithms. Variables of the best-performing algorithm (LR) were selected through stepwise use of Akaike's information criterion (AIC). The constructed prediction model was compared with the methylation biomarkers and the Mayo Clinic model using the non-parametric approach of DeLong et al. with the area under a receiver operator characteristic curve (AUC) analysis. RESULTS: A prediction model was finally constructed based on three DNA methylation biomarkers and one radiological characteristic for identifying malignant from benign PNs. The developed prediction model achieved an AUC value of 0.951 in malignant PNs diagnosis, significantly higher than the three DNA methylation biomarkers (0.912, 95% CI:0.843-0.958, p = 0.013) or Mayo Clinic model (0.823, 95% CI:0.739-0.890, p = 0.001). Validation of the prediction model in the testing cohort of 100 subjects with PNs confirmed the diagnostic value. CONCLUSION: We have shown that integrating DNA methylation biomarkers and radiological characteristics could more accurately identify lung cancer in subjects with CT-found PNs. The prediction model developed in our study may provide clinical utility in combination with LDCT to improve the over-all diagnosis of lung cancer.

DNA methylation of PTGER4 in peripheral blood plasma helps to distinguish between lung cancer, benign pulmonary nodules and chronic obstructive pulmonary disease patients.

BACKGROUND/INTRODUCTION: In contrast to patients who present with advanced stage lung cancer and associated poor prognosis, patients with early-stage lung cancer may be candidates for curative treatments. The results of the NELSON lung cancer screening trial are expected to stimulate the development and implementation of a lung cancer screening strategy in most countries. Widespread use of chest computed tomography scans will also result in the detection of solitary pulmonary nodules. Because reliable biomarkers to distinguish between malignant and benign lesions are lacking, tissue-based histopathological diagnostics remain the gold standard. In this study, we aimed to establish a test to assess the predictive ability of DNA hypermethylation of SHOX2 and PTGER4 in plasma to discriminate between patients with 1.) lung cancer, 2.) benign lesions, and 3.) patients with chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: We retrospectively analysed SHOX2 and PTGER4 methylation in 121 prospectively collected plasma samples of patients with lung cancer (group 1A), benign lesions (group 1B), and COPD without nodules (group 2). RESULTS: PTGER4 DNA hypermethylation was more frequently observed in patients with lung cancer than in controls (p = 0.0004). Results remained significant after correction for tumour volume, smoking status, age, and eligibility for the NELSON trial. CONCLUSIONS: Detection of methylated PTGER4 in plasma DNA may serve as a biomarker to support clinical decision-making in patients with pulmonary lesions at lung cancer screening in high-risk populations. Further exploration in prospective studies is warranted.

Homeobox A5 and A9 expression and beta-thalassemia.

Background and aim: The pathogenesis of ß-thalassemia has been attributed to ineffective erythropoiesis. The function of Hox genes in normal haematopoiesis has been widely studied using gene expression analysis. The aim of this study is to evaluate the expression of HoxA9, and HoxA5 genes in beta-thalassemia.Materials and methods: Children with thalassemia major, thalassemia intermediate, and age and sex-matched healthy controls (n = 50/group) were enrolled. Detection of HoxA5 and HoxA9 mRNA expression was performed by real-time polymerase chain reaction (RT-PCR).Results: Expression of HoxA9 increased in a direct linear trend (median 0.5 in controls, 2.4 in intermediate disease, 4.1 in major disease, p = 0.001) and generally correlated with the red cell count, haematocrit, ferritin and levels of beta-globin. In those with thalassemia major, the relative change of HoxA9 was linked to transfusion history, the white blood cell count, ferritin, and beta-globin (all r > 0.5, p < 0.001). Levels of HoxA9 were superior to HoxA5 in differentiating controls from thalassemia intermedia, whilst both differentiated major from the intermediate disease.Conclusion: This study highlights the importance of HoxA genes in early identification of patients, at high risk of developing complications, as it allows specific measures to delay the progression of the disease. HoxA gene expression is a promising diagnostic and prognostic marker in patients with ß-thalassemia.

Evaluation of blood gene expression levels in facioscapulohumeral muscular dystrophy patients.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the expression of DUX4 in skeletal muscles. A number of therapeutic approaches are being developed to antagonize the events preceding and following DUX4 expression that leads to muscular dystrophy. Currently, the possibility to evaluate treatment response in clinical trials is hampered by the lack of objective molecular biomarkers connecting the disease cause to clinical performance. In this study we employed RNA-seq to examine gene expression in PAXgene tubes obtained from two independent cohorts of FSHD patients. Analysis of gene expression profiles did not lead to the identification of genes or pathways differentially expressed in FSHD patients, or associated with disease severity. In particular, we did not find evidence that the DUX4 and PAX7 signatures were differentially expressed. On the other hand, we were able to improve patient classification by including single genes or groups of genes in classification models. The best classifier was ROPN1L, a gene known to be expressed in testis, coincidentally the typical location of DUX4 expression. These improvements in patient classification hold the potential to enrich the FSHD clinical trial toolbox.

Detection of Promoter DNA Methylation in Urine and Plasma Aids the Detection of Non-Small Cell Lung Cancer.

PURPOSE: Low-dose CT screening can reduce lung cancer-related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation-based detection of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: This nested case-control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation-specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42). RESULTS: DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively. CONCLUSIONS: DNA methylation-based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

Treatment Response Monitoring in Patients with Advanced Malignancies Using Cell-Free SHOX2 and SEPT9 DNA Methylation in Blood: An Observational Prospective Study.

Patients with incurable cancer usually receive palliative treatment with significant toxicity and limited efficacy. Methylation analysis of circulating cell-free DNA (ccfDNA) in blood from cancer patients represents a promising approach for minimally invasive, real-time monitoring of treatment response. Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) methylation was analyzed in N = 8865 malignant and N = 746 normal adjacent tissues across 33 different malignancies from The Cancer Genome Atlas. Furthermore, we performed quantitative SHOX2 and SEPT9 ccfDNA methylation analysis in plasma obtained before and consecutively during treatment from prospectively enrolled N = 115 patients with various advanced cancers. SHOX2 and/or SEPT9 hypermethylation in malignant tissues is present in various carcinomas, sarcoma, melanoma, brain tumors, mesothelioma, and hematopoietic malignancies. Among the prospectively enrolled cancer patients, 61% (70/115) of patients had a baseline-positive blood cumulative ccfDNA methylation score (CMS) and were eligible for response monitoring. Dynamic changes of CMS during treatment were strongly associated with treatment response. A CMS increase indicated response up to 80 days before conventional monitoring. SHOX2 and SEPT9 ccfDNA methylation represents a pan-cancer biomarker and has the potential to be a powerful tool for monitoring treatment response in patients with solid tumors and lymphomas. The early identification of nonresponders might allow for a timely change of treatment regimen.

Detection of aberrant methylation of HOXA9 and HIC1 through multiplex MethyLight assay in serum DNA for the early detection of epithelial ovarian cancer.

Accumulated evidence revealed that aberrant CpG island hypermethylation plays an important role in carcinogenesis which can serve as a promising target for molecular detection in body fluids. Despite a myriad of attempts to diagnose ovarian cancer (OC) at an early stage, this clinical aim remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant DNA methylation patterns in circulating DNA provide highly specific cancer signals. In our study, we establish a novel panel of methylation-specific genes for the development of a TaqMan based qPCR assay to quantify methylation levels. We analyzed promoter methylation of homeobox A9 (HOXA9) and hypermethylated in cancer 1 (HIC1) quantitatively in 120 tissue samples and in 70 matched serum cell-free DNA (CFDNA) of cancerous and noncancerous samples by MethyLight assay. HOXA9 and HIC1 methylation occurred in 82.3 and 80.0% of OC tissue samples in singleplex assay, thereby confirming that methylation was highly cancer-specific. When either or both gene promoter showed methylation, the sensitivity was 88.2% with a specificity of 88.6% in tissue samples. The combined sensitivity for this novel marker panel in serum CFDNA was 88.9% (area under the curve [AUC] = 0.95). In contrast, no hypermethylation was observed in serum from matched cancer-free control women. Our results confirm the elevated performance of novel epigenetic marker panel (HOXA9 and HIC1) when analyzed in tissue and matched serum samples. Our findings reveal the potential of this biomarker panel as a suitable diagnostic serum biomarker for early screening of OC.

Correlation of miR-181a and three HOXA genes as useful biomarkers in acute myeloid leukemia.

INTRODUCTION: MiR-181a is a small, noncoding RNA that plays important roles in the pathogenesis and prognosis of acute myeloid leukemia (AML). A group of HOXA genes, including HOXA7, HOXA9, and HOXA11, has been established as an independent predictor for AML prognosis. In this study, we aimed to investigate the association between miR-181a and HOXA7, HOXA9, and HOXA11 and explore their roles in predicting prognosis in AML. PATIENTS AND METHODS: Bone marrow samples of 46 untreated AML patients and 9 healthy donors were collected. Mononuclear cells were purified using density-gradient centrifugation in Ficoll, and quantitative real-time PCR was used to detect miR-181a and HOXA gene expression level. RESULTS: HOXA7, HOXA9, and HOXA11 were negatively correlated with miR-181a, and their expression levels varied among AML subtypes, karyotypes, and risk status. Higher miR-181a and lower HOXA gene expressions were significantly associated with lower risk status and better response to chemotherapy. CONCLUSION: In our study, we found miR-181a expression was negatively correlated with three HOXA genes and they were associated with AML risk status and prognosis in granulocytic AML. It further supported that miR-181a could be a useful marker for AML prognosis and possibly worked by regulating HOXA gene clusters.

Study of expression of genes potentially responsible for reduced fitness in patients with myotonic dystrophy type 1 and identification of new biomarkers of testicular function.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by trinucleotide CTG expansion in DMPK gene, often affecting the neighboring genes. Endocrine system is involved, resulting in hypogonadism and reproductive abnormalities, but molecular mechanisms underlying the reduced fertility observed in DM1 are very complex and partially unknown. To better characterize these mechanisms, an analysis of sperm parameters and anti-Müllerian hormone (AMH) values was performed in 20 DM1 patients. About 50% of them showed hypoposia and azoospermia; the remaining, despite an adequate volume of ejaculate, had oligo-astheno-teratozoospermia. Interestingly, the lowest AMH levels better correlated with the main sperm alterations. The pattern of expression of DMPK, SIX5, and RSPH6A genes, evaluated by quantitative reverse transcription polymerase chain reaction, showed a substantial reduction of the expression in both peripheral blood and in seminal plasma of patients, compared to controls. An impairment of testis-specific RSPH6A protein expression and localization was observed in sperm protein extracts by WB analysis and in isolated spermatozoa by immunofluorescence. These results support the hypothesis that CTG expansion also affects the expression of neighboring genes and contributes to gonad defects observed in DM1, suggesting the possibility of using them as markers for normal fertility in humans.

[DNA methylation detection of SHOX2 and PTGER4 in plasma contributes to differential diagnosis of pulmonary nodule patients].

Objective To investigate the clinical significance of plasma cell free DNA (cfDNA) methylation in short stature homeobox 2 (SHOX2) and prostaglandin E receptor 4 (PTGER4) for diagnosing pulmonary nodule patients. Methods We collected 10 mL venous blood from patients. And the plasma DNA was extracted. Real-time quantitative PCR was performed to amplify the DNA after bisulfite conversion. Valid Ct values were input into specialized software to analyze the methylation status of SHOX2 and PTGER4. Histological classification of lung malignant tissues was carried out by immunohistochemistry. Results The 22 of 57 patients were positive and 35 were negative for SHOX2 and PTGER4 DNA methylation detection. Computed tomography (CT) indicated that 31 of 57 patients were diagnosed with pulmonary nodules, among which 19 patients were positive for DNA methylation; 3 of 16 patients with inflammation on CT were positive for DNA methylation; 10 patients with normal or ground glass CT images were negative for DNA methylation. Significant differences in SHOX2 and PTGER4 DNA methylation were observed in the patients with different CT findings. The highest positive rate of CT nodular lesions was 61.3%. The 20 patients with pulmonary nodules were pathologically diagnosed with lung cancer, of which 18 were positive for SHOX2 and PTGER4 DNA methylation, with a positive rate of 90%. Only 1 case of benign pulmonary nodules was positive. Significant difference in SHOX2 and PTGER4 methylation status was observed between benign and malignant pulmonary nodules. The positive rate of SHOX2 and PTGER4 methylation were both 100% in squamous cell lung carcinoma and small cell lung carcinoma, while 75% in adenocarcinoma. Conclusion SHOX2 and PTGER4 methylation detection in blood plasma has certain value in the early diagnosis of lung cancer and can be a complementary tool of CT in diagnosing pulmonary nodule patients.

Normal Growth despite Combined Pituitary Hormone Deficiency.

BACKGROUND: The paradox of normal growth despite a lack of growth hormone (GH) is an unexplained phenomenon described in some pathological (sellar, suprasellar, and hypothalamic disorders) and overgrowth syndromes. It has been suggested that the paradoxical growth is due to other GH variants, GH-like moieties, prolactin, insulin, insulin-like growth factors (IGFs), and unidentified serum factors or growth mechanisms. The objective of this study was to determine the mechanism underlying this normal growth without GH. CASE DESCRIPTION: We describe here growth, hormonal, and genetic analyses for an adolescent boy with panhypopituitarism who achieved an adult height above his genetic potential. RESULTS: Normal growth was observed despite low serum GH, IGF-I, IGF-II, IGF binding protein 3 (IGFBP-3) and acid labile subunit (ALS) concentrations, but the IGF-II/IGFBP-3 molar ratio was slightly high. Panhypopituitarism was associated with a heterozygous missense mutation of HESX1, with variable penetrance in heterozygous relatives. Exome analysis detected heterozygous missense mutations of various genes involved in intracellular signaling pathways. The growth-promoting activity of the patient's serum was unable to induce AKT phosphorylation in the MCF-7 cell line. CONCLUSION: The high IGF-II/IGFBP-3 molar ratio was not the cause of the sustained high growth velocity, due to the low affinity of IGF-II for IGF type 1 receptor. The key finding was the HESX1 mutation, as similar cases have been described before, suggesting a common mechanism for growth without GH. However, the variable penetrance of this variant in heterozygous relatives suggests that modifier genes or mechanisms involving combinations with mutations of other genes involved in intracellular signaling pathways might be responsible.

Increased serum FGF2 levels in first-episode, drug-free patients with schizophrenia.

Preclinical and clinical studies suggest that brain-derived neurotrophic factor and nerve growth factor are involved in the pathogenesis of schizophrenia (SCZ). However, the roles of other neurotrophic factors in SCZ remain unclear. The aim of this study was to investigate the blood levels of FGF2 and ADNP in first-episode, drug-free SCZ patients compared with healthy control subjects. 20 SCZ patients, and 20 age and sex matched controls were recruited in this study. Serum FGF2 and ADNP protein levels were measured by ELISA assay, and the results showed that FGF2 levels were significantly increased in patients with SCZ when compared with controls, whereas ADNP protein levels did not significantly associated with SCZ. However, we found that blood ADNP mRNA levels were significantly increased in the patients with SCZ when compared with controls. In addition, subgroup analyses suggested that FGF2 levels were significantly increased in female patients of SCZ, but not in male patients of SCZ. Correlation analyses suggested that age and disease severity (PANSS score) did not have moderating effects on the serum FGF2 levels. Taken together, our results for the first time demonstrated that blood FGF2 was up-regulated in first-episode, drug free-SCZ patients, therefore enhancing the knowledge of neurotrophic factor profile in patients with SCZ.

Evaluation of the HOXA11 level in patients with lung squamous cancer and insights into potential molecular pathways via bioinformatics analysis.

BACKGROUND: This study was carried out to discover the underlying role that HOXA11 plays in lung squamous cancer (LUSC) and uncover the potential corresponding molecular mechanisms and functions of HOXA11-related genes. METHODS: Twenty-three clinical paired LUSC and non-LUSC samples were utilized to examine the level of HOXA11 using quantitative real-time polymerase chain reaction (qRT-PCR). The clinical significance of HOXA11 was systematically analyzed based on 475 LUSC and 18 non-cancerous adjacent tissues from The Cancer Genome Atlas (TCGA) database. A total of 102 LUSC tissues and 121 non-cancerous tissues were available from Oncomine to explore the expressing profiles of HOXA11 in LUSC. A meta-analysis was carried out to further assess the differential expression of HOXA11 in LUSC, including in-house qRT-PCR data, expressing data extracted from TCGA and Oncomine databases. Moreover, the enrichment analysis and potential pathway annotations of HOXA11 in LUSC were accomplished via Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The expression of hub genes and according correlations with HOXA11 were assessed to further explore the biological role of HOXA11 in LUSC. RESULTS: HOXA11 expression in LUSC had a tendency to be upregulated in comparison to adjacent non-cancerous tissues by qRT-PCR. TCGA data displayed that HOXA11 was remarkably over-expressed in LUSC compared with that in non-LUSC samples, and the area under curves (AUC) was 0.955 (P < 0.001). A total of 1523 co-expressed genes were sifted for further analysis. The most significant term enriched in the KEGG pathway was focal adhesion. Among the six hub genes of HOXA11, including PARVA, ILK, COL4A1, COL4A2, ITGB1, and ITGA5, five (with the exception of COL4A1) were significantly decreased compared with the normal lung tissues. Moreover, the expression of ILK was negatively related to HOXA11 (r = - 0.141, P = 0.002). CONCLUSION: High HOXA11 expression may lead to carcinogenesis and the development of LUSC. Furthermore, co-expressed genes might affect the prognosis of LUSC.

Aberrant DNA methylation at HOXA4 and HOXA5 genes are associated with resistance to imatinib mesylate among chronic myeloid leukemia patients.

BACKGROUND: Imatinib mesylate is a molecularly targeted tyrosine kinase inhibitor drug. It is effectively used in the treatment of chronic myeloid leukemia (CML) patients. However, development of resistance to imatinib mesylate as a result of BCR-ABL dependent and BCR-ABL independent mechanisms has emerged as a daunting problem in the management of CML patients. Between these mechanisms, BCR-ABL independent mechanisms are still not robustly understood. AIM: To investigate the correlation of HOXA4 and HOXA5 promoter DNA hypermethylation with imatinib resistance among CML patients. METHODS AND RESULTS: Samples from 175 Philadelphia positive CML patients (83 good response and 92 BCR-ABL non-mutated imatinib resistant patients) were subjected to Methylation Specific High Resolution Melt Analysis for methylation levels quantification of the HOXA4 and HOXA5 promoter regions. Receiver operating characteristic curve analysis was done to elucidate the optimal methylation cut-off point followed by multiple logistic regression analysis. Log-Rank analysis was done to measure the overall survival difference between CML groups. The optimal methylation cut-off point was found to be at 62.5% for both HOXA4 and HOXA5. Chronic myeloid leukemia patients with ≥63% HOXA4 and HOXA5 methylation level were shown to have 3.78 and 3.95 times the odds, respectively, to acquire resistance to imatinib. However, overall survival of CML patients that have ≤62% and ≥ 63% methylation levels of HOXA4 and HOXA5 genes were found to be not significant (P-value = 0.126 for HOXA4; P-value = 0.217 for HOXA5). CONCLUSION: Hypermethylation of the HOXA4 and HOXA5 promoter is correlated with imatinib resistance and with further investigation, it could be a potential epigenetic biomarker in supplement to the BCR-ABL gene mutation in predicting imatinib treatment response among CML patients but could not be considered as a prognostic marker.

Determinants of platelet count in pediatric patients with congenital cyanotic heart disease: Role of immature platelet fraction.

OBJECTIVES: Congenital heart defects are common noninfectious causes of mortality in children. Bleeding and thrombosis are both limiting factors in the management of such patients. We assessed the frequency of thrombocytopenia in pediatric patients with congenital cyanotic heart disease (CCHD) and evaluated determinants of platelet count including immature platelet fraction (IPF) and their role in the pathogenesis of thrombocytopenia. METHODS: Forty-six children and adolescents with CCHD during pre-catheter visits were studied; median age was 20.5 months. Complete blood count including IPF as a marker of platelet production and reticulated hemoglobin content (RET-He) as a marker of red cell production and iron status were done on Sysmex XE 2100 (Sysmex, Japan). C-reactive protein, prothrombin time (PT), Activated partial thromboplastin time (APTT) were also assessed. RESULTS: Thrombocytopenia was found in 6 patients (13%). PT was prolonged (P = .016) and IPF was significantly higher in patients with thrombocytopenia compared with patients with normal platelet count (14.15 ± 5.2% vs 6.68 ± 3.39%; P = .003). Platelet count was negatively correlated with IPF while significant positive correlations were found between IPF and hemoglobin, red blood cells (RBCs) count, hematocrit (Hct), PT, reticulocytes count, and immature reticulocyte fraction. CONCLUSIONS: We suggest that elevated IPF in CCHD patients with thrombocytopenia may denote peripheral platelets destruction as an underlying mechanism. Hemoglobin level, RBCs count, Hct, and RET-He were not significant determinants for platelet count in CCHD.

DNA Methylation Analysis of Free-Circulating DNA in Body Fluids.

Circulating cell-free DNA in body fluids is an analyte of great interest in basic and clinical research. The analyses of DNA methylation and hydroxymethylation patterns in body fluids might allow one to determine the certain state of a disease, in particular of cancer. DNA methylation biomarkers in liquid biopsies, i.e. blood plasma samples, may help optimizing personalized therapy for individual patients. DNA methylation analyses of specific loci usually require a bisulfite conversion of the DNA, which requires a sufficiently high amount of DNA at the appropriate concentration. However, free-circulating DNA is generally low concentrated. Therefore, high volumes of body fluids need to be analyzed. This high volume needs to be reduced in order to facilitate the bisulfite conversion. In addition, disease-related free-circulating DNA is even less abundant than normal DNA in the total amount of free-circulating DNA. Accordingly, analytical and pre-analytical methods are needed, which permit an accurate and sensitive quantification of single methylated DNA copies in the presence of unmethylated DNA in abundance.This protocol describes two methods for DNA enrichment from body fluids: DNA extraction by means of magnetic beads and polymer-mediated enrichment of DNA. Subsequent bisulfite conversion is achieved by means of a high-speed conversion protocol. Adaptions of the workflow required for the analysis of hydroxymethylation via oxidation 5-hydroxymethylcytosines to 5-formylcytosines prior to the bisulfite conversion are introduced. A quantitative real-time PCR based on the methylation-specific and HeavyMethyl PCR methodologies is introduced. This qPCR assay allows for an accurate and sensitive quantification of single copies of the DNA methylation biomarkers SHOX2 and SEPT9 in blood plasma. Specific issues regarding the analysis of body fluids and respective trouble shooting approaches are discussed.

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

BACKGROUND: SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. METHODS: Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9/SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. RESULTS: Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61-63% sensitivity/90-92% specificity (SEPT9/training), 53-57% sensitivity/87-90% specificity (SHOX2/training), and 64-65% sensitivity/90-91% specificity (mean SEPT9/SHOX2 /training). Results were confirmed in a testing cohort with 54-56% sensitivity/88-90% specificity (SEPT9/testing), 43-48% sensitivity/93-95% specificity (SHOX2/testing), and 49-58% sensitivity/88-94% specificity (mean SEPT9/SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals (SEPT9: AUCtraining = 0.79-0.80; AUCtesting = 0.74-0.75; SHOX2: AUCtraining = 0.78-0.81, AUCtesting = 0.77-0.79; mean SEPT9/SHOX2 : AUCtraining = 0.81-0.84, AUCtesting = 0.80). The accurate prediction of overall survival was possible with all three algorithms (training cohort: HR SEPT9 = 1.23-1.90, HR SHOX2 = 1.14-1.85, HRmeanSEPT9/SHOX2 =1.19-1.89 ; testing cohort: HR SEPT9 =1.22-1.67, HR SHOX2 = 1.15-1.71, HRmeanSEPT9/SHOX2 = 1.12-1.77). CONCLUSION: The concordant clinical performance based on different quantification algorithms allows for the application of various diagnostic platforms for the analysis of ccfDNA methylation biomarkers.