Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

<p>Abstract</p> <p>Background</p> <p>DNA methylation in the <it>SHOX2 </it>locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with <it>SHOX2 </it>gene expression and/or copy number alterations. An amplification of the <it>SHOX2 </it>gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.</p> <p>Methods</p> <p><it>SHOX2 </it>expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect <it>SHOX2 </it>DNA methylation levels. <it>SHOX2 </it>expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.</p> <p>Results</p> <p>A hypermethylation of the <it>SHOX2 </it>locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the <it>SHOX2 </it>gene showed no difference.</p> <p>Conclusions</p> <p>Frequent gene amplification correlated with hypermethylation of the <it>SHOX2 </it>gene locus. This concerted effect qualifies <it>SHOX2 </it>DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.</p

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

<p>Abstract</p> <p>Background</p> <p>This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.</p> <p>Methods</p> <p>Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.</p> <p>Results</p> <p>Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).</p> <p>Conclusions</p> <p>Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.</p

DNA Methylation of the Homeobox Genes PITX2 and SHOX2 Predicts Outcome in Non-small-cell Lung Cancer Patients

Biomarkers that facilitate prediction of disease progression in lung cancer patients might be clinically valuable in optimizing individualized therapy. In this study, the ability of the DNA methylation biomarkers PITX2 and SHOX2 to predict disease outcome in lung cancer patients has been evaluated. Quantitative, methylation-specific (HeavyMethyl), real-time polymerase chain reaction assays were used to measure DNA methylation of PITX2 and SHOX2 in bisulfite-converted DNA from formalin-fixed, paraffin-embedded tissues from 474 non-small-cell lung cancer patients. In univariate Cox Proportional Hazard analysis, high methylation of SHOX2 and PITX2 was a significant predictor of progression-free survival [SHOX2: n=465, hazard ratio (HR)=1.395 (1.130 to 1.721), P=0.002; PITX2: n=445, HR=1.312 (1.059 to 1.625), P=0.013]. Patients with low methylation of either PITX2 and/or SHOX2 (n=319) showed a significantly higher risk of disease progression as compared with patients with higher methylation of both genes [n=126; HR=1.555 (1.210 to 1.999), P=0.001]. This was particularly true for the subgroup of patients receiving no adjuvant radiotherapy or chemotherapy [n=258, HR=1.838 (1.252 to 2.698), P=0.002]. In multivariate analysis, both biomarkers added significant independent prognostic information to pT, pN, pM, and grade. Another interesting finding of this study was that SHOX2 and PITX2 DNA methylation was shown to be inversely correlated with TTF1 (also known as NKX2-1) expression (PITX2: P=0.018, SHOX2: P<0.001). TFF1 expression was previously found to be associated with improved survival in the same patient cohort. DNA methylation of PITX2 and SHOX2 is an independent prognostic biomarker for disease progression in non-small-cell lung cancer patients

Quantification of cell-free mSHOX2 Plasma DNA for therapy monitoring in advanced stage non-small cell (NSCLC) and small-cell lung cancer (SCLC) patients.

PURPOSE:Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response. MATERIAL AND METHODS:In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC (34 stage IV, 2 stage IIIB) of whom 34 received standard platinum-based chemo/radiotherapy and two were treated with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHOX2) in plasma before and during therapy until re-staging. The mSHOX2 analysis was blinded with respect to the clinical data making it an observational study. RESULTS:According to the re-staging of 31 first-line patients, 19 patients were classified as non-responders while 12 patients were in the responder group. We observed a tight correlation between radiological data and the change of plasma mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power for both patient groups already one week after therapy start (AUC 0.844). Additionally, a Kaplan-Meier and Cox Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p ≤ 0.001 (hazard ratio 11.08) providing some evidence for mSHOX2 also being a predictive marker. CONCLUSION:The longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment

Clinical data of first-line patients responding to the therapy.

<p>Ten of the patients were male and two were female. The median age of this patient group is 61.5 years. Patient UKH 031 demonstrated an EGFR mutation and was treated with TKI.</p><p>Clinical data of first-line patients responding to the therapy.</p

Trend curves for patients responding (black curves) and not responding (gray curves) to the therapy.

<p>The patients included in this figure are limited to the ones with a baseline mSHOX2 value of at least 1% PMR. The first blood draw (x = 0) is the point of diagnosis, i.e. before treatment and defines the baseline methylation of SHOX2. For the first eight blood draws Bonferroni corrected p-values from unpaired two sample Wilcox tests are given at the bottom.</p

Cox Proportional Hazards and Kaplan-Meier survival analysis of all 36 patients.

<p>The median plasma <i>mSHOX2</i> value at baseline was 2.88% PMR, while the median one week after therapy start was 2.16% PMR.</p

Clinical data of second-line patients.

<p>In this group were four male and one female patients. The only female patient in this group (UKH 001) did respond to therapy. The median age of this patient group is 64 years. Patient UKH 010 demonstrated an EGFR mutation and was treated with TKI.</p><p>Clinical data of second-line patients.</p

Glyoxylate, a New Marker Metabolite of Type 2 Diabetes

Type 2 diabetes (T2D) is characterized by a variety of metabolic impairments that are closely linked to nonenzymatic glycation reactions of proteins and peptides resulting in advanced glycation end-products (AGEs). Reactive aldehydes derived from sugars play an important role in the generation of AGEs. Using metabolite profiling to characterize human plasma from diabetic versus nondiabetic subjects we observed in a recent study that the reactive aldehyde glyoxylate was increased before high levels of plasma glucose, typical for a diabetic condition, could be measured. Following this observation, we explored the relevance of increased glyoxylate in diabetic subjects and in diabetic C57BLKS/J-Leprdb/db-/- mice in the pathophysiology of diabetes. A retrospective study using samples of long-term blood donors revealed that glyoxylate levels unlike glucose levels became significantly elevated up to 3 years prior to diabetes diagnosis (difference to control P=0.034). Elevated glyoxylate levels impact on newly identified mechanisms linking hyperglycemia and AGE production with diabetes-associated complications such as diabetic nephropathy. Glyoxylate in its metabolic network may serve as an early marker in diabetes diagnosis with predictive qualities for associated complications and as potential to guide the development of new antidiabetic therapies