{"id":1149,"date":"2017-04-06T20:53:23","date_gmt":"2017-04-06T18:53:23","guid":{"rendered":"http:\/\/www.newslab.sk\/2017\/04\/06\/performance-of-a-dna-methylation-marker-panel-using-liquid-based-cervical-scrapes-to-detect-cervical-cancer-and-its-precancerous-stages\/"},"modified":"2017-10-03T07:55:40","modified_gmt":"2017-10-03T05:55:40","slug":"performance-of-a-dna-methylation-marker-panel-using-liquid-based-cervical-scrapes-to-detect-cervical-cancer-and-its-precancerous-stages","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/performance-of-a-dna-methylation-marker-panel-using-liquid-based-cervical-scrapes-to-detect-cervical-cancer-and-its-precancerous-stages\/","title":{"rendered":"Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages"},"content":{"rendered":"
*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf \r\nattachment at the end of the paper. \r\n\r\n<\/span><\/strong><\/pre>\n
Intr<\/strong>oduction<\/strong><\/p>\n
Cervical cancer is still among the most frequent cancers in women world-wide (Torre et al., 2015). \u00a0With the availability of screening programs, however, cervical cancer incidence and mortality \u00a0have markedly decreased, especially in developed \u00a0countries (Anttila et al., 2009). \u00a0The effects of the \u00a0cytology-based diagnostics \u2013 the so-called Pap test, \u00a0the most prominent screening \u00a0tool \u00a0applied \u00a0even \u00a0nowadays \u2013 have, however, levelled-off \u00a0the last\u00a0 decade, mainly because of the limited \u00a0sensitivity for precancerous lesions, as well as limited participation of the\u00a0 women. On the\u00a0 other \u00a0hand, \u00a0limited specificity of the Pap test also leads to over-diagnosis and over-treatment, mainly among young \u00a0women. Therefore al- ternative screening tools, \u00a0which \u00a0may \u00a0lead \u00a0to an\u00a0 overcome of these limitations of cytology \u00a0are \u00a0discussed since several years.<\/p>\n
Testing for the human papillomaviruses (hrHPV) that evoke \u00a0cervical \u00a0cancer improves the sensitivity of screening (Ronco et al., 2014). In some countries (e.g. the Netherlands, USA) HPV testing has \u00a0already \u00a0been implemented in screening. Infection \u00a0with one of the high-risk HPV strains is the prerequisite for the development of cervical \u00a0cancer. Therefore, HPV screening has \u00a0high sensitivity. It lacks, \u00a0however, specificity, since \u00a0most women infected with HPV will clear \u00a0such an infection without symptoms. Therefore, HPV-based cervical cancer screening only makes sense with the availability of triage \u00a0methods that \u00a0allow the detection of precancerous lesions and \u00a0cancer cases among women tested HPV-positive (Wentzensen et al., 2015).<\/p>\n
In this\u00a0 context, hypermethylation of certain DNA regions during the course of carcinogenesis may provide a promising tool for triage of a highly sensitive screening, which finds virtually all disease cases, but lacks specificity, as is the case if testing for HPV infection (Lorincz et al., 2013; Wentzensen et al., 2015). We have previously shown that detection of a DNA hypermethylation marker \u00a0panel \u00a0consisting of the five marker regions DLX1, ITGA4, RXFP3, SOX17, and ZNF671 may be a useful \u00a0tool for triaging HPV-positive women (Hansel et al., 2014). Here we show that a molecular diagnostic test based on the marker \u00a0regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and \u00a0ZNF671, termed GynTect, which\u00a0 received CE IVD mark in October \u00a02015, can \u00a0easily \u00a0be adapted for liquid-based cytology samples triage.<\/p>\n
 <\/p>\n
Materials and <\/strong>Methods<\/strong><\/p>\n
 <\/p>\n
Informed consent<\/em><\/strong><\/p>\n
All samples utilized in this study were collected only after informed consent of patient was gained.<\/p>\n
 <\/p>\n
P<\/em><\/strong>atient samples<\/em><\/strong><\/p>\n
Residual \u00a0liquid-based cytology \u00a0(LBC) samples from both, routine \u00a0cervical \u00a0cancer screening as well as more \u00a0accurate diagnostics for further \u00a0triage \u00a0following an abnormal cytology result \u00a0(Pap III or higher) were used for the study. \u00a0The collection consisted of 60 screening samples from women with normal cytology (PapI), and 45 screening and triage samples from women with histopathology diagnosis CIN1\/2 (14 sam- ples), \u00a0CIN3 (26 samples), and \u00a0cervical \u00a0cancer (5 samples). All samples were collected in PreservCyt medium (Hologic).<\/p>\n
For all samples cytology results were available. For the samples with abnormal Pap smear finding histopathology results were available, classifying them into the different CIN stages.<\/p>\n
 <\/p>\n
Sample <\/em><\/strong>pr<\/em><\/strong>eparation<\/em><\/strong><\/p>\n
For sample preparation and \u00a0lysis the \u00a0LBC samples were vortexed for a few seconds, and \u00a01 ml of each sample were immediately transferred into 1.5-ml microcentrifuge vials. Cellular material was pelleted by centrifugation at 10,000 x g for 5 min, and the supernatant was \u00a0removed carefully \u00a0by pipetting. Pellets were \u00a0then \u00a0resuspended in 40 \u00b5l of sample lysis buffer (GynTect, oncgnotics), and incubated at 60\u00b0C for 30 min at 1,000 \u00a0rpm \u00a0in a thermoshaker (Thermomixer, Ep- pendorf).<\/p>\n
 <\/p>\n
Bisulfite treatment and marker methylation analysis<\/strong><\/p>\n
Bisulfite \u00a0treatment of cervical \u00a0samples \u00a0was performed using \u00a0the \u00a0EpiTect \u00a0Fast \u00a0Bisulfite \u00a0Kit (Qiagen) following \u00a0the supplier\u2019s manual. 40 \u00b5l of the cervical \u00a0sample was \u00a0directly used for bisulfite \u00a0treatment without \u00a0prior DNA isolation. After elution \u00a0in 20 \u00b5l Elution Buffer, 70 \u00b5l of water \u00a0was \u00a0added, and 10 \u00b5l of the diluted DNA were used for each single reaction in the GynTect real-time \u00a0methylation-specific PCR (qM- SP) assay as described in the manual of the GynTect kit. The qMSPs \u00a0were run on a ABI 7500 Real-Time PCR System (Life technologies, Thermo \u00a0Scientific). Ct values for each marker and \u00a0each internal \u00a0control \u00a0were recorded, and \u00a0their validity was controlled by comparing the melting \u00a0curve characteristics of each PCR fragment produced with corresponding positive controls. For such \u00a0positive controls, which were in- cluded in each PCR run, DNA known to be methylated in the marker \u00a0regions was \u00a0used. A no template control using \u00a0water \u00a0as template was \u00a0also \u00a0included in each qMSP \u00a0run. \u00a0For evaluation, the \u00a0difference of the \u00a0Ct values of each marker with the \u00a0internal \u00a0control \u00a0ACHE was calculated. qMSPs \u00a0for ASTN1, DLX1, ITGA4, RXFP3 ans SOX17 yielding a difference between sample and internal control \u00a0\u2264 9.0 were scored pos- itive, for ZNF671 \u226410 was \u00a0scored positive. A GynTect assay was \u00a0scored positive, \u00a0if the \u00a0sum \u00a0of the \u00a0factors attributed to each marker \u00a0was 0.5 or higher (Table 1)<\/em><\/strong>.<\/p>\n
 <\/p>\n
Results<\/strong><\/p>\n
For assessing methylation of the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, ZNF671 as well as the two internal marker \u00a0regions ACHE and IDS an intercalating dye-based qMSP assay was performed for each of the 105 patient samples \u00a0included in this \u00a0study. \u00a0The results obtained for the \u00a060 samples from patients with a cytology \u00a0result \u00a0Pap \u00a0I were the basis for setting the\u00a0 delta \u00a0Ct value limit \u2264 9 for all markers but ZNF671, for which the delta Ct value limit was set to \u2264 10.<\/p>\n
To be scored valid, the Ct value for the control \u00a0marker \u00a0ACHE had to be below 32. At these settings, 59 of the 60 Pap I samples were scored negative for the GynTect test. This evaluation was then the basis for scoring the data \u00a0obtained for the 45 CIN1+ samples.<\/p>\n
All five carcinomas included in the study were scored GynTect-positive. Of the \u00a026 samples with histopathology-confirmed CIN3, 16 (= 61.5%) turned out to be GynTect-positive, whereas of the 14 CIN1\/2 samples seven (= 50%) were GynTect-positive (Figure 1A)<\/em><\/strong>. When related to cytology findings, the following results were obtained for the CIN samples: of the \u00a017 samples scored Pap \u00a0III or Pap \u00a0IIID in cytology, seven (= 41%) turned out to be GynTect-positive; of the 23 Pap IVa samples, 16 (= 69.6%) were GynTect-positive (Figure 1B)<\/em><\/strong>.<\/p>\n
 <\/p>\n
Discussion<\/strong><\/p>\n
In previous studies \u00a0we \u00a0have \u00a0shown that \u00a0hypermethylation of CpG islands in proximity \u00a0to the \u00a0genes DLX1<\/em>, ITGA4<\/em>, RXFP3<\/em>, SOX17<\/em>, and ZNF671 <\/em>correlated with the presence of precancerous cervical lesions and cervical cancer (Hansel et al., 2014). \u00a0The molecular diagnostic test GynTect based on these results allows the detection of these marker \u00a0regions in cervical smears collected in the denaturing specimen trans- port medium (STM), which \u00a0is originally used for QIAGEN\u2019s DIGENE HPV test. Utilization of this \u00a0medium has, however, its limitations, the most important being that from STM only molecular test formats can be performed. In contrast, cervical smear material collected in liquid-based cytology \u00a0media can \u00a0be used more flexibly. As a main \u00a0advantage, the \u00a0cellular material preserved in this medium can be used for cytology as well as molecular biology tests. This enables the performance of triage tests from the same sample as the initial screening test, a feature which increasingly is demanded as prerequisite for diagnostics.<\/p>\n
In this study we evaluated whether our molecular diagnostic test GynTect is suitable for using \u00a0residual material from liquid-based cytology samples and such \u00a0fulfils this prerequisite. GynTect provides the possibility \u00a0to test if a woman who\u00a0obtained an abnormal cytology finding in the Pap smear and\/ or a positive HPV test result, has \u00a0a precancerous lesion \u00a0that requires follow-up and treatment. For this purpose we used samples for which the cytology \u00a0findings \u00a0and, for all Pap-abnormal \u00a0samples, the \u00a0histopathology results were \u00a0available for comparison. GynTect showed an excellent performance, since for all 105 samples valid test results could be obtained. The results obtained for the \u00a0two \u00a0internal \u00a0markers that \u00a0are tested with each patient sample demonstrate this. \u00a0In fact, the Ct values for these two internal \u00a0markers obtained for all 105 \u00a0LBC samples are \u00a0much \u00a0lower\u00a0 than \u00a0those obtained for samples collected in STM, indicating better preservation of the DNA in these LBC samples. Due to the improved performance, a threshold for the marker \u00a0Ct values in relation to the controls was \u00a0set. \u00a0Using a delta \u00a0Ct threshold of 9 for the five markers ASTN1, DLX1, ITGA4, RXFP3, and SOX17 as well as a delta \u00a0Ct threshold of 10 for ZNF671, of the \u00a060 samples with a normal cytology, Pap I, only one sample yielded a GynTect-positive result, implying that \u00a0the test has a very good specificity within this \u00a0group. \u00a0A larger \u00a0number of such \u00a0samples will definitely have to be examined to confirm \u00a0these results and this very high specificity of the test among healthy women.<\/p>\n
As expected, all 5 cervical cancer samples included in the study were \u00a0detected by GynTect, and \u00a0that \u00a0by at least four GynTect markers. This very high sensitivity for cancer cas- es was already \u00a0shown previously (Hansel et al., 2014). \u00a0A detection rate \u00a0of >60% among the CIN3 samples examined also \u00a0confirms results obtained in previous studies (Hansel et al., 2014). \u00a0It is well-known \u00a0that \u00a0not \u00a0all CIN3 lesions proceed to cervical \u00a0cancer (McCredie \u00a0et \u00a0al., 2008), \u00a0although these \u00a0high-grade lesions \u00a0are\u00a0 \u00a0considered as precancer- ous \u00a0stages. In several observational studies CIN2\/3\u00a0 shortterm \u00a0regression rates around 30% were reported (Trimble et al., 2005, \u00a02010, \u00a02015; Grimm et al., 2012). \u00a0Very recently, Loopik et al. (2016) \u00a0have demonstrated in a retrospective study \u00a0that \u00a0in women < 25 years \u00a0the regression rate \u00a0of CIN2 lesions was as high as 71% (150 \u00a0of 211 \u00a0women followed\u00a0after CIN2 diagnosis), and the overall progression rate in this study \u00a0was very low (15%).<\/p>\n
The data \u00a0show that the GynTect score is related to the severity of the lesion \u00a0confirmed by histopathology. In fact, the higher the CIN grade, the more GynTect markers are positive in the \u00a0LBC samples. The only GynTect-positive CIN1 case, however, had a score of 1.2, so five of the six markers were positive, with rather low delta Ct values. In cytology this case was graded Pap IVa, which might imply that the biopsy in this case was \u00a0not taken \u00a0at the punctum maximum of the lesion. Altogether, the correlation between cytology finding and GynTect result \u00a0was even higher.<\/p>\n
In conclusion, GynTect, a test which provides a triage \u00a0option for either HPV-based or cytology-based cervical \u00a0cancer screening, shows excellent results if performed on cervical scrape material in liquid-based cytology \u00a0media, \u00a0a prerequisite for employing such \u00a0a test in new screening programs.<\/p>\n
 <\/p>\n
REFERENCES<\/strong>
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\nof VGX-3100, a therapeutic synthetic DNA vaccine targeting
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\nphase 2b trial. Lancet 2015; 386: 2078-88.
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\nS49-55.<\/p>\n
 <\/p>\n
 <\/p>\n","protected":false},"excerpt":{"rendered":"
*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper. Introduction Cervical cancer is still among the most frequent cancers in women world-wide (Torre et al., 2015). \u00a0With the availability of screening programs, however, cervical cancer incidence and mortality \u00a0have<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[290],"tags":[615,616,613,614],"class_list":["post-1149","post","type-post","status-publish","format-standard","hentry","category-genetics","tag-biomarkers","tag-cervical-cancer","tag-dna-methylation","tag-human-papillomavirus-hpv","typ_clanku-original-work"],"acf":{"abstrakt":"
Aim: <\/em><\/strong>An essential event in early carcinogenesis is the hypermethylation of so-called CpG islands, which are predominantly located in promoter\/5\u2019 regions of genes in the human genome. Specific patterns of hypermethylation may thus be indicative for carcinogenesis and provide tools for diagnostics. In the current study the performance of a panel of six DNA methylation marker regions for the detection of cervical precancerous lesions and cancer was assessed using cervical scrapes from corresponding patients.<\/p>\n
Methods: <\/em><\/strong>A series of cervical scrapes from women with cervical cancer (n=5), cervical intraepithelial neoplasia grade 3 (CIN3) (n=26) or CIN1\/2 (n=14), and women with normal cytology (n=60) were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Methylation analysis was done using the GynTect\u00ae assay.<\/p>\n
Results: <\/em><\/strong>All samples from women with cervical cancer (5\/5) were scored positive for the methylation assay. Of the CIN3 cases, 62% (16\/26), of the CIN1\/2 cases 50% (7\/14) were positive for the assay. Only 1.7% of the cy- tology-normal samples (1\/60) were positive for the methylation assay. Overall, the number of methylated mark- er regions increased proportionally to the lesion severity.<\/p>\n
Conclusion: <\/em><\/strong>DNA methylation analysis of ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1\/2 lesions, whereas the detection rate among cytology-normal samples is extraordinarily low. Thus, the GynTect\u00ae assay based on detection of these six methylation markers may provide an excellent tool within cervical cancer screening.<\/p>\n
K<\/strong>e<\/strong>ywords<\/strong>: DNA methylation; human Papillomavirus (HPV); biomarkers; cervical cancer<\/p>\n","casopis":[{"ID":735,"post_author":"7","post_date":"2017-04-06 13:21:01","post_date_gmt":"2017-04-06 11:21:01","post_content":"
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