Haukeline H. Volders

Assessment of gene promoter hypermethylation for detection of cervical neoplasia

Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation‐specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot‐study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 patients diagnosed with cervical cancer (20 squamous cell carcinomas and 10 adenocarcinomas) and 19 women with histologically normal cervices. The scraped cells were used for determination of promoter hypermethylation by QMSP for 12 genes and for morphological assessment. Overall, CALCA, DAPK, ESR1, TIMP3, APC and RAR‐β2 promoters were significantly more often hypermethylated in cancers than in controls, while adenocarcinomas were more often hypermethylated above the highest control ratio for APC, TIMP3 and RASSF1A promoters. Combining 4 genes (CALCA, DAPK, ESR1 and APC) yielded a sensitivity of 89% (with all adenocarcinomas identified), equal to cytomorphology (89%) and high‐risk human papilloma virus (Hr‐HPV; 90%). The 4‐gene QMSP proved theoretically superior to cytomorphology as well as Hr‐HPV in specificity (100% vs. 83 and 68%, respectively), because cytology identified 3 controls as moderate or severe dyskaryosis and 6 controls were positive for Hr‐HPV. In conclusions, QMSP of 4 gene promoters combined appears to have comparable sensitivity and potentially better specificity in comparison to “classic” cytomorphological assessment and Hr‐HPV detection. QMSP holds promise as a new diagnostic tool for both squamous cell carcinoma and adenocarcinoma of the cervix.

Involvement of the TGF-beta and beta-Catenin Pathways in Pelvic Lymph Node Metastasis in Early-Stage Cervical Cancer

Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer. Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways. Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P < 0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR: 0.20, 95% CI: 0.06–0.66) and the β-catenin pathway (p120 positivity) to N+ (OR: 1.79, 95%CI: 1.05–3.05). Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated noncanonical β-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer. Clin Cancer Res; 17(6); 1317–30. ©2011 AACR.

Methylation markers for CCNA1 and C13ORF18 are strongly associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings.

Purpose: Recently, we reported 13 possible cervical cancer–specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance. Experimental Design and Results: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high. Conclusion: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3000–7)

Detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages, a feasibility study

OBJECTIVE To explore the feasibility of DNA methylation analysis for the detection of cervical neoplasia in self-obtained cervico-vaginal lavages. METHODS Lavages collected by a self-sampling device and paired cervical scrapings were obtained from 20 cervical cancer patients and 23 patients referred with an abnormal cervical smear (15 with high-grade cervical intraepithelial neoplasia (CIN2+) and 8 without CIN). All lavages and scrapings were analyzed by liquid based cytology (LBC), Hybrid Capture II (HC-II) for hr-HPV DNA detection and by DNA methylation analysis (JAM3, TERT, EPB41L3 and C13ORF18). Concordance between lavages and scrapings was measured by Cohens Kappa (k). RESULTS In lavages and scrapings from cervical cancer patients (n=20), methylation analysis was positive in 19 (95%) and 19 (95%), HC-II in 16 (80%) and 15 (75%) and LBC in 15 (75%) and 19 (95%), respectively. In lavages and scrapings from CIN2+ patients (n=15), methylation analysis was positive in 10 (67%) and 12 (80%), HC-II in 15 (100%) and 15 (100%) and LBC in 11 (73%) and 12 (80%), respectively. Concordance between cervical scrapings and lavages (n=43) was for LBC k=0.522 (p<0.001), hr-HPV testing k=0.551 (p<0.001) and DNA methylation analysis k=0.653 (p<0.001). CONCLUSIONS DNA methylation analysis in cervico-vaginal lavages obtained by a self-sampling device is feasible and its diagnostic performance appears to be at least comparable to the detection of cervical neoplasia by cytomorphology and hr-HPV. Our pilot study suggests that detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages warrants exploration of its use in large prospective studies.

l-Arginine deficiency causes airway hyperresponsiveness after the late asthmatic reaction

Peroxynitrite has been shown to be crucially involved in airway hyperresponsiveness (AHR) after the late asthmatic reaction (LAR). Peroxynitrite production may result from simultaneous synthesis of nitric oxide (NO) and superoxide by inducible NO-synthase (iNOS) at low l-arginine concentrations. l-Arginine availability to iNOS is regulated by its cellular uptake, which can be inhibited by eosinophil-derived polycations and by arginase, which competes with iNOS for the common substrate. Using a guinea pig model of allergic asthma, we investigated whether aberrant l-arginine homeostasis could underlie peroxynitrite-mediated AHR after the LAR. After the LAR, arginase activity in the airways and eosinophil peroxidase release from bronchoalveolar lavage cells were increased. These changes were associated with a 2.0-fold AHR to methacholine as measured in isolated perfused tracheal preparations. AHR was reduced by exogenous l-arginine administration. Moreover, both the arginase inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA) and the polycation antagonist heparin normalised airway responsiveness. These effects were reversed by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME), indicating that both agents reduced AHR by restoring bronchodilating NO production. In conclusion, in allergen-challenged guinea pigs, the AHR after the LAR is caused by arginase- and polycation-induced attenuation of l-arginine availability to iNOS, which may switch the enzyme to simultaneous production of superoxide and NO, and, consequently, peroxynitrite.

Discovery of DNA methylation markers in cervical cancer using relaxation ranking

BackgroundTo discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.AimTo apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.ResultsThe application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue.ConclusionThe application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.

An Overview of Innovative Techniques to Improve Cervical Cancer Screening

Although current cytomorphology-based cervical cancer screening has reduced the incidence of cervical cancer, Papsmears are associated with high false positive and false negative rates. This has spurred the search for new technologies to improve current screening. New methodologies are automation of Pap-smear analysis, addition of new biological or molecular markers to traditional cytology or using these new markers to replace the current screening method. In this overview we will summarize data on cervical cancer epidemiology and etiology and the current cervical cancer screening approach. Available data on new screening approaches, such as quantitative cytochemistry, detection of loss of heterozygosity (LOH) and hypermethylation analysis will be reviewed.We discuss the potential of these approaches to replace or augment current screening. When available, data on cost– effectiveness of certain approaches will be provided. In short, Human Papillomavirus (HPV) DNA detection stands closest to implementation in nation-wide screening programs of all markers reviewed. However, specificity is low in women aged <35 years and the psychological effects of knowledge of HPV positivity in absence of cervical (pre) malignant disease are important drawbacks. In our opinion the results of large clinical trials should be awaited before proceeding to implement HPV DNA detection. New technologies based on molecular changes associated with cervical carcinogenesis might result in comparable sensitivity, but improved specificity. Hypermethylation analysis is likely to be more objective to identify patients with high grade squamous intra-epithelial lesions (HSIL) or invasive cancer with a higher specificity than current cytomorphology based screening.

Identification of inguinofemoral lymph node metastases by methylation markers in vulvar cancer.

OBJECTIVE Lymph node status in early-stage vulvar cancer can be accurately assessed by the sentinel-node (SN) procedure. Molecular techniques, such as DNA-methylation assay, might improve SN assessment. In this study, we selected methylation markers for vulvar cancer and determined if these methylation markers were suitable for lymph node assessment. METHODS We performed methylation specific PCR on DNA isolated from primary tumors, metastatic lymph nodes, and negative lymph nodes from twenty vulvar cancer patients using the following genes: P16INK4a, MGMT, TWIST1, CADM1, TERT, and TFPI2. For P16INK4a and MGMT immunohistochemistry was performed on primary tumors and metastatic lymph nodes in order to explore intratumor heterogeneity in gene expression patterns. RESULTS TERT was methylated in all vulvar cancers, P16INK4a in 13/20, TFPI2 in 12/20, CADM1 in 11/20, MGMT in 9/20, and TWIST1 in 7/20. A panel of three methylation markers (P16INK4a, TERT and TFPI2) reached a sensitivity of 67% and specificity of 100% for detection of metastatic lymph nodes. Immunohistochemistry showed intratumor heterogeneity for expression of P16INK4a and MGMT in respectively 55% and 45% of primary tumors. CONCLUSIONS Our study shows methylation for one or more methylation markers in all vulvar cancers. Despite a specificity of 100% our panel of three methylation markers had only moderate sensitivity for metastatic lymph node detection, thereby limiting its applicability for lymph node assessment. Intratumor heterogeneity for expression of P16INK4a and MGMT may reflect intratumor heterogeneity for methylation patterns and thereby in general explain the moderate sensitivity of our marker panel for detection of metastases.

Mo1797 Cigarette Smoke is an Environmental Factor That Reduces Innate Immune Functions Especially in Individuals With the CD-Associated Risk Variant ATG16L1-T300A

Chronic granulomatous disease (CGD) is an X-linked or autosomal recessive genetic defect resulting in defective microbial killing and recurrent bacterial and fungal infections or inflammation, mainly in the respiratory and gastrointestinal tracts. Symptoms and findings of CGD overlap with inflammatory bowel diseases (IBD), and anti-microbial serologic responses were reported. However, in Crohns disease (CD), serologic responses reflect loss of tolerance towards microorganisms and glycans. Anti-glycan antibodies (AGA) directed against the glycans laminaribioside, chitobioside, mannobioside and mannan further characterize CD patients. Aim: To assess the titers and prevalence of AGA in CGD patients. Methods: Serum samples were collected from well characterized CGD patients, patients with other innate immune defects (hyper IgE/ Hermanski-Pudlak syndrome, HIE/HPS), CD and healthy controls. AGA were assessed using ELISA (IBDx, Glycominds Ltd, Israel) and compared to previously determined serologic responses against the microbial antigens OmpC, I2, and CBir1 performed on the same serum samples. Results: Sixty one CGD patients (48 males, age at sample collection 24.4±1.4 years, 69% x-linked 91 phox mutation, 36% with colitis), 7 patients with HIE/HPS , 397 CD patients and 12 healthy controls were recruited. gASCA titers and prevalence were significantly higher in CGD, compared to HIE/HPS (129±5 EU, 95% vs. 35±12EU, 43%, p<0.001) and CD patients (p<0.001). Interestingly, AGA prevalence in CGD was roughly half (34-41%) and titer increase above cut-off was lower, compared to the prevalence reported for OmpC, I2 and CBir1 (69-75%) and their respective titer increases in the same samples. Two or more positive AGA were detected in 67% CGD, 22% CD, 6% HIE/HPS patients, and 7% healthy controls, respectively. gASCA was significantly lower, while ANCA higher, in CGD patients younger, compared to older than 18 years (p= 0.02, p=0.056, respectively). No differences in the prevalence of AGA were observed whether CGD patients had IBD (n=22) or not. Conclusions: CGD patients have a significantly higher serologic response against microorganisms and glycans compared to CD and other innate immune defects controls. Serologic responses against ASCA increase with age suggesting that the mechanism may be recurrent exposure. The discordant prevalence and titers of the antibody response towards microbial vs. glycan antigens in CGD may suggest lower immunogenicity of the glycans. The finding of AGA characteristic of CD in CGD, supports the notion that an innate immune defect underlies CD pathogenesis.