Dickkopf-3 as a new potential marker for neoangiogenesis in colorectal cancer: expression in cancer tissue and adjacent non-cancerous tissue.

Gene expression of Dickkopf-3 (Dkk-3) has been shown to be upregulated in tumor endothelium of colorectal cancer (CRC). For the first time, we analyzed Dkk-3 protein expression in CRC and its potential as a marker for neoangiogenesis. We used tissue microarrays (TMAs) to investigate Dkk-3 in microvessels of 403 CRC samples, 318 appropriate adjacent non-cancerous samples and 127 normal colorectal samples. Of cancer samples with CD31-positive microvessels, 67.7% were positive for Dkk-3. Dkk-3 staining was demonstrated in endothelial cells of all microvessels in nearly all cases. Dkk-3-positive samples showed a higher mean microvessel count than did Dkk-3-negative samples (P=0.001). Dkk-3 expression increased with rising numbers of microvessels per sample (P<0.0001). In adjacent samples with CD31-positive microvessels, 56% were Dkk-3-positive in all microvessels. Similar to cancer samples, Dkk-3-positive adjacent samples had a higher mean microvessel count than did Dkk-3-negative samples (P<0.0001), and Dkk-3 expression also increased with rising numbers of microvessels (P<0.0001). All microvessels in normal mucosa samples were negative for Dkk-3. Dkk-3 can be considered a putative pro-angiogenic protein in neovascularization and may possibly be a marker for neoangiogenesis in CRC. Further investigations will elucidate whether Dkk-3 is a target structure for novel therapies.

Circulating cell-free DNA in plasma of locally advanced rectal cancer patients undergoing preoperative chemoradiation: a potential diagnostic tool for therapy monitoring.

Circulating cell-free DNA opens up an interesting field for therapy monitoring, in particular during multimodal therapy protocols. The objective of this proof of principle study was to evaluate whether the amount of circulating plasma DNA has the potential to serve as a marker for therapy monitoring during the treatment course of locally advanced rectal cancer patients. We especially focused on kinetics of circulating DNA to assess whether variances in kinetics have the potential to discriminate between therapy responders and nonresponders. The amount of circulating DNA in plasma of rectal cancer patients undergoing preoperative chemoradiation was determined using real-time PCR before chemoradiation, after the end of chemoradiation and at the end of treatment. The study population was divided into responders (ypT0-T2 stage) and nonresponders (ypT3-T4 stage). Both groups showed comparable median plasma DNA values before and after the end of chemoradiation. At the end of treatment responders showed a further decrease in circulating DNA, whereas in nonresponders the circulating DNA manifestly increased (P = 0.006). This study demonstrates that circulating DNA in plasma of rectal cancer patients undergoing preoperative chemoradiation might serve as a surrogate marker to discriminate between responders and nonresponders. Therefore, we hypothesize that quantification of plasma DNA could be of use as an easily accessible tool for therapy monitoring in these patients.

DNA methylation in colorectal cancer--impact on screening and therapy monitoring modalities?

Colorectal cancer (CRC) is a common malignancy. It arises from benign neoplasms and evolves into adenocarcinomas through a stepwise histological progression sequence, proceeding from either adenomas or hyperplastic polyps/serrated adenomas. Genetic alterations have been associated with specific steps in this adenoma-carcinoma sequence and are believed to drive the histological progression of CRC. Recently, epigenetic alterations (especially DNA methylation) have been shown to occur in colon polyps and CRC. The aberrant methylation of genes appears to act together with genetic alterations to drive the initiation and progression of colon polyps to CRC. DNA methylation changes have been recognized as one of the most common molecular alterations in human tumors, including CRC. Because of the ubiquity of DNA methylation changes and the ability to detect methylated DNA in several body fluids (blood, stool), this specifically altered DNA may serve, on the one hand, as a possible new screening marker for CRC and, on the other hand, as a tool for therapy monitoring in patients having had neoplastic disease of the colorectum. As many CRC patients present with advanced disease, early detection seems to be one of the most important approaches to reduce mortality. Therefore, an effective screening test would have substantial clinical benefits. Furthermore, early detection of progression of disease in patients having had CRC permits immediate commencement of specific treatment regimens (e.g. curative resection of liver and lung metastases) and probably longer survival and better quality of life.

DNA methylation in serum of breast cancer patients: an independent prognostic marker.

Changes in the status of DNA methylation are one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated whether aberrant DNA methylation in patient pretherapeutic sera is of prognostic significance in breast cancer. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 39 genes in a gene evaluation set, consisting of 10 sera from metastasized patients, 26 patients with primary breast cancer, and 10 control patients. To determine the prognostic value of genes identified within the gene evaluation set, we finally analyzed pretreatment sera of 24 patients having had no adjuvant treatment (training set) to determine their prognostic value. An independent test set consisting of 62 patients was then used to test the validity of genes and combinations of genes, which in the training set were found to be good prognostic markers. In the gene evaluation set we identified five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A). In the training set, patients with methylated serum DNA for RASSF1A and/or APC had the worst prognosis (P < 0.001). This finding was confirmed by analyzing serum samples from the independent test set (P = 0.007). When analyzing all 86 of the investigated patients, multivariate analysis showed methylated RASSF1A and/or APC serum DNA to be independently associated with poor outcome, with a relative risk for death of 5.7. DNA methylation of particular genes in pretherapeutic sera of breast cancer patients, especially of RASSF1A/APC, is more powerful than standard prognostic parameters.

Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.

We have generated DNA methylation profiles of 148 human breast tumors and found significant differences in hormone receptor (HR) status between clusters of DNA methylation profiles. Of 35 DNA methylation markers analyzed, the ESR1 gene, encoding estrogen receptor alpha, proved to be the best predictor of progesterone receptor status, whereas methylation of the PGR gene, encoding progesterone receptor, was the best predictor of estrogen receptor status. ESR1 methylation outperformed HR status as a predictor of clinical response in patients treated with the antiestrogen tamoxifen, whereas promoter methylation of the CYP1B1 gene, encoding a tamoxifen- and estradiol-metabolizing cytochrome p450, predicted response differentially in tamoxifen-treated and nontamoxifen-treated patients. High levels of promoter methylation of the ARHI gene, encoding a RAS-related small G-protein, were strongly predictive of good survival in patients who had not received tamoxifen therapy. Our results reveal an as yet unrecognized degree of interaction between DNA methylation and HR biology in breast cancer cells and suggest potentially clinically useful novel DNA methylation predictors of response to hormonal and non-hormonal breast cancer therapy.

hCDC4 gene mutations in endometrial cancer.

Cyclin-dependent kinase 2 activated by cyclin E is involved in the initiation of DNA replication and other S phase functions. Consistent with this role, cyclin E protein accumulates at the G1-S phase transition and declines during early S phase. This profile of expression is the result of periodic transcription and ubiquitin-mediated proteolysis directed by SCF(hCdc4). However, in many types of human tumors cyclin E protein is elevated and deregulated relative to the cell cycle by an unknown mechanism. Here, we show that the F-box protein hCdc4 that targets cyclin E to the SCF (Skp1-Cull-F-box) protein ubiquitin ligase is mutated in at least 16% of human endometrial tumors. Mutations were found either in the substrate-binding domain of the protein or at the amino terminus, suggesting a critical role for the region of hCdc4 upstream of the F-box. hCDC4 gene mutations were accompanied by loss of heterozygosity and correlated with aggressive disease. The hCDC4 gene is localized to chromosome region 4q32, which is deleted in over 30% of human tumors. Our results show that the hCDC4 gene is mutated in primary human tumors and suggest that it may function as a tumor suppressor in the genesis of many human cancers.

DNA hypomethylation and ovarian cancer biology.

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromere-adjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5' gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from non-neoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.

Cyclin E dysregulation and chromosomal instability in endometrial cancer.

Deregulation of cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), has been associated with a broad spectrum of human malignancies. Yet the mechanism linking abnormal cyclin E expression to carcinogenesis is largely unknown. The gene encoding the F-box protein hCdc4, a key component of the molecular machinery that targets cyclin E for degradation, is frequently mutated in endometrial cancer, leading to deregulation of cyclin E expression. Here we show that hCDC4 gene mutation and hyperphosphorylation of cyclin E, a parameter that usually correlates with hCDC4 mutation, have a strong statistically significant association with polypoidy and aneuploidy in endometrial cancer. On the contrary, elevated expression of cyclin E by itself was not significantly correlated with polyploidy or aneuploidy when tumors of similar grade are evaluated. These data suggest that impairment of cell cycle regulated proteolysis of cyclin E may be linked to carcinogenesis by promoting genomic instability.

Methylation changes in faecal DNA: a marker for colorectal cancer screening?

DNA methylation is a common molecular alteration in colorectal cancer cells. We report an assessment of faecal DNA from patients with colorectal cancer and controls to determine the feasibility, sensitivity, and specificity of this approach. By use of MethyLight analysis of faecal DNA from three independent sets of patients, we identified SFRP2 methylation as a sensitive single DNA-based marker for identification of colorectal cancer in stool samples (sensitivity 90% [CI 56-100] and specificity 77% [46-95] in the training set [n=23]; sensitivity 77% [46-95] and specificity 77% [46-95] in an independent test set [n=26]). Whether a combination of genetic and epigenetic markers will identify colorectal cancer at an early stage remains to be shown.

Circulating nucleic acids in plasma or serum (CNAPS) as prognostic and predictive markers in patients with solid neoplasias.

It is now widely accepted that there is a need for the development of molecular markers of cancer that can be used for clinical prognostication and monitoring. Approximately a decade ago tumor-derived circulating nucleic acids in the plasma or serum (CNAPS) of cancer patients were introduced as a noninvasive tool for cancer detection. This review focuses on the various types of CNAPS of patients with solid neoplasias (genetic alterations in circulating DNA, microsatellites, methylated DNA, viral DNA, nucleosomes, mitochondrial DNA and cell-free mRNA) and their putative potential as prognostic or predictive parameter or even as a tool for therapy monitoring during follow-up. Additionally, this review aims to point out the difference between a prognostic and a predictive factor in patient bloodstream. However, with rapid technical improvement and well-designed studies we conclude that the next years will see CNAPS analysis integrated in the prognostication and monitoring of cancer patients, thus producing more specific treatment regimens for patients with various stages of neoplastic disease and ultimately longer survival and better quality of life.

DNA methylation in serum and tumors of cervical cancer patients.

PURPOSE: Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease. EXPERIMENTAL DESIGN: The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique. RESULTS: Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P = 0.04) and overall survival (P = 0.02) in comparison with patients with methylated MYOD1. CONCLUSIONS: To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

Analysis of aberrant DNA methylation and human papillomavirus DNA in cervicovaginal specimens to detect invasive cervical cancer and its precursors.

PURPOSE: Cancer of the uterine cervix is an important cause of death in women worldwide. Pap smears as a tool for screening decreased the incidence and mortality of cervical cancer dramatically. This proof of principle study aimed to develop a potential tool for cervical screening using a test that can be applied by patients without visiting a physician and to increase the coverage rate, especially of the high-risk population with low socioeconomic status. EXPERIMENTAL DESIGN: Human papillomavirus (HPV) DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens of 13, 31, and 11 patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer, respectively, collected on a tampon, was performed using PCR-based methods to detect invasive cervical cancer and study whether these changes are already present in the precursor lesions. RESULTS: High-risk HPV DNA was present in 68 and 82% of patients with high-grade SIL and invasive cervical cancer. DNA methylation of the 11 genes tested increased with severity of the cervical lesion. Unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes was able to predict the presence of invasive cervical cancers: one of the two clusters formed contained 9 of 11 invasive cervical cancers, as well as two high-grade SILs. CONCLUSIONS: HPV DNA and DNA methylation analyzed in cervicovaginal specimens are able to predict invasive cervical cancers. To detect all high-grade SILs when applying this test, genes that become methylated earlier throughout cervical carcinogenesis have to be defined.

Methylated DNA collected by tampons--a new tool to detect endometrial cancer.

This proof of principle study aimed to define a new and simple strategy for detection of endometrial cancer using epigenetic markers. We investigated DNA isolated from vaginal secretion collected from tampon for aberrant methylation of five genes (CDH13, HSPA2, MLH1, RASSF1A, and SOCS2) using MethyLight in 15 patients with endometrial cancer and 109 patients without endometrial cancer. All endometrial cancer patients revealed three or more methylated genes, whereas 91% (99 of 109) of the patients without endometrial cancer had no or fewer than three genes methylated in their vaginal secretion. The methods developed in this study provide the basis for a prospective clinical trial to screen asymptomatic women who are at high risk for endometrial cancer.

Human papillomavirus DNA in sera of cervical cancer patients as tumor marker.

Persistent infection with human papillomavirus (HPV) has been widely recognized to induce carcinoma of the uterine cervix. Viral DNA has been documented to occur as tumor DNA in the circulation of patients with primary tumors caused by viral infection. The aim of this study was to evaluate the utility of serum HPV DNA as tumor marker in cervical cancer patients. Sera taken from 94 cervical cancer patients at the date of diagnosis and follow-up serum samples from 24 patients were examined for HPV DNA using PCR enzyme immunoassay. Serum samples taken at the date of diagnosis were HPV DNA positive in 45% of all investigated samples. Sera which were HPV DNA positive at the time of diagnosis became and also remained negative after primary treatment in patients without recurrence or persistent disease. HPV DNA was positive up to 423 days before the time of clinical diagnosis of recurrence in 10 out of 13 cases (median 72 days, range 0-423 days). Serum HPV DNA seems to reflect biological activity of the tumor. Our results indicate that serum HPV DNA might be a useful additional marker for early detection of recurrence in cervical cancer patients.

A DNA methylation pattern similar to normal tissue is associated with better prognosis in human cervical cancer.

Promoter hypermethylation has been recognized to play an important role in carcinogenesis. We analyzed the methylation status of 25 genes in 14 normal cervical tissue specimens and in 65 tissue specimens from cervical cancer patients using the MethyLight technique. Most of the analyzed genes have been shown to be methylated in various cancers. RB1 was never methylated in any analyzed cervical tissue. DNA methylation status of the remaining 24 genes in every tissue sample was subsequently analyzed using unsupervised agglomerative hierarchical cluster analysis to group specimens and CpG regions. We observed four clusters. All normal cervical tissue specimens were grouped together in one cluster. Neither grade, nor histological type nor age demonstrated any significant association with clusters formed. Interestingly, statistically significantly less patients whose tumor DNA methylation pattern clustered together with normal cervical tissue died within our observation period, as compared to those patients out of the three remaining clusters (P < 0.03) Cervical cancer patients, whose DNA methylation pattern clustered together with normal cervical tissue revealed a strong trend to better survival (P = 0.066) compared to patients grouped in the remaining cluster. This study shows for the first time that working solely with DNA methylation pattern a subgroup of cervical cancer patients can be defined that demonstrated strong similarity to non-neoplastic probands and had a better prognosis.

Methylated DNA as a possible screening marker for neoplastic disease in several body fluids.

Early detection appears to be one of the most important approaches to reducing mortality caused by neoplasia. Changes in DNA methylation have been recognized as one of the most common molecular alterations in human tumors. Due to the ubiquity of DNA methylation changes and the possibility to detect methylated DNA in several body fluids, this specifically altered DNA may serve as a potential new screening marker for neoplastic disease.

Prognostic DNA methylation marker in serum of cancer patients.

Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasia. Recent demonstrations of tumor-derived methylated DNA in the blood stream of cancer patients allow the use of these epigenetic markers for risk assessment in cancer patients. We were interested in evaluating the prognostic value of several methylated genes in the serum of cancer patients. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 215 serum samples from patients with cervical (n = 93) or breast cancer (n = 122) for DNA methylation changes. In cervical cancer, hypermethylation of three genes (MYOD1, CDH1, and CDH13) in pretreatment sera was statistically significantly associated with a poorer disease outcome. Additionally, for the first time we used a so-called gene evaluation set to identify the most important DNA methylation changes in the serum of breast cancer patients from a long list of candidate genes. In the gene evaluation set, we detected five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A) using our criteria for further analysis. Finally, two of the evaluated genes (APC and RASSF1A) proved to be independent prognostic parameters in breast cancer patients. In summary, we detected several prognostic DNA methylation markers in the serum of cervical and breast cancer patients. This finding indicates great potential for the use of these epigenetic markers in clinical, routine risk assessment in patients with various malignancies.

14-3-3 sigma and p53 expression in gastric cancer and its clinical applications.

14-3-3 sigma (σ) induces G2 arrest enabling the repair of damaged DNA. The function of 14-3-3 σ is frequently lost in tumor cells, indicating a potential tumor suppressor function. The purpose of this study was to evaluate the prognostic value of 14-3-3 σ expression in human gastric cancer. 14-3-3 σ expression was analyzed by immunohistochemistry in 157 tumor samples of patients, who underwent resection for gastric cancer. Since 14-3-3 σ is involved in the p53 network, p53 expression was detected in parallel and correlated with 14-3-3 σ. 14-3-3 σ was found to be overexpressed in 75 (47.8%) of 157 cases, the overexpression rate of p53 protein was 27.4%. 14-3-3 σ overexpression was statistically significantly associated with pT-stage (p=0.041) pN-stage (p=0.015) and UICC-stage (p=0.019) and showed a borderline significance with Lauren classification (p=0.057). Univariate survival calculations revealed a coexistent 14-3-3 σ and p53 overexpression as a significant predictor of disease-free survival. Multivariate analysis did not unfold 14-3-3 as an independent prognostic factor for disease-free survival and overall survival. Concomitant 14-3-3 σ and p53 overexpression in tumor cells of patients with gastric cancer identifies a population of patients with relatively unfavorable prognosis.

SFRP2 methylation in fecal DNA--a marker for colorectal polyps.

INTRODUCTION: DNA methylation of secreted frizzled-related proteins (SFRPs) can be detected in colorectal cancer (CRC) tissue, in tissue of adenomas, and in aberrant crypt foci, whereas in normal colorectal mucosa tissue, SFRP genes are unmethylated. Recently, our study group was able to demonstrate SFRP2 methylation as the most sensitive single DNA-based marker in stool for identification of CRC. The purpose of this study was to clarify whether SFRP2 methylation in fecal DNA can be found in stool of individuals with hyperplastic and adenomatous colorectal polyps. MATERIALS AND METHODS: Patients who were diagnosed with colorectal polyps or showed negative colonoscopy were included in this study. DNA from stool samples was isolated. SFRP2 methylation was assessed by means of MethyLight. RESULTS: Stool samples from 68 individuals were checked for DNA content; 23% of the samples (6 of 26) from healthy controls, 46% of the samples (6 of 13) from patients with hyperplastic polyps, and 45% of the samples (13 of 29) from patients with adenomas were positive for human DNA. SFRP2 methylation in stool samples was found in none of the healthy controls, in 33% (2 of 6) patients with hyperplastic polyps, and in 46% (6 of 13) patients with adenomas. Statistical analysis revealed that the frequency of SFRP2 methylation increased significantly (P=0.028) from healthy controls to patients with hyperplastic polyps and to patients with adenomas. CONCLUSIONS: In the current study, we report for the first time that SFRP2 methylation in fecal DNA increases significantly from healthy controls to patients with hyperplastic polyps and to patients with adenomas. SFRP2 methylation may serve as a marker for molecular stool-based adenoma and CRC screening.