*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).  With the availability of screening programs, however, cervical cancer incidence and mortality  have markedly decreased, especially in developed  countries (Anttila et al., 2009).  The effects of the  cytology-based diagnostics – the so-called Pap test,  the most prominent screening  tool  applied  even  nowadays – have, however, levelled-off  the last  decade, mainly because of the limited  sensitivity for precancerous lesions, as well as limited participation of the  women. On the  other  hand,  limited specificity of the Pap test also leads to over-diagnosis and over-treatment, mainly among young  women. Therefore al- ternative screening tools,  which  may  lead  to an  overcome of these limitations of cytology  are  discussed since several years.

Testing for the human papillomaviruses (hrHPV) that evoke  cervical  cancer improves the sensitivity of screening (Ronco et al., 2014). In some countries (e.g. the Netherlands, USA) HPV testing has  already  been implemented in screening. Infection  with one of the high-risk HPV strains is the prerequisite for the development of cervical  cancer. Therefore, HPV screening has  high sensitivity. It lacks,  however, specificity, since  most women infected with HPV will clear  such an infection without symptoms. Therefore, HPV-based cervical cancer screening only makes sense with the availability of triage  methods that  allow the detection of precancerous lesions and  cancer cases among women tested HPV-positive (Wentzensen et al., 2015).

In this  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  panel  consisting of the five marker regions DLX1, ITGA4, RXFP3, SOX17, and ZNF671 may be a useful  tool for triaging HPV-positive women (Hansel et al., 2014). Here we show that a molecular diagnostic test based on the marker  regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and  ZNF671, termed GynTect, which  received CE IVD mark in October  2015, can  easily  be adapted for liquid-based cytology samples triage.

Materials and Methods

Informed consent

All samples utilized in this study were collected only after informed consent of patient was gained.

Patient samples

Residual  liquid-based cytology  (LBC) samples from both, routine  cervical  cancer screening as well as more  accurate diagnostics for further  triage  following an abnormal cytology result  (Pap III or higher) were used for the study.  The 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),  CIN3 (26 samples), and  cervical  cancer (5 samples). All samples were collected in PreservCyt medium (Hologic).

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.

Sample preparation

For sample preparation and  lysis the  LBC samples were vortexed for a few seconds, and  1 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  removed carefully  by pipetting. Pellets were  then  resuspended in 40 µl of sample lysis buffer (GynTect, oncgnotics), and incubated at 60°C for 30 min at 1,000  rpm  in a thermoshaker (Thermomixer, Ep- pendorf).

Bisulfite treatment and marker methylation analysis

Bisulfite  treatment of cervical  samples  was performed using  the  EpiTect  Fast  Bisulfite  Kit (Qiagen) following  the supplier’s manual. 40 µl of the cervical  sample was  directly used for bisulfite  treatment without  prior DNA isolation. After elution  in 20 µl Elution Buffer, 70 µl of water  was  added, and 10 µl of the diluted DNA were used for each single reaction in the GynTect real-time  methylation-specific PCR (qM- SP) assay as described in the manual of the GynTect kit. The qMSPs  were run on a ABI 7500 Real-Time PCR System (Life technologies, Thermo  Scientific). Ct values for each marker and  each internal  control  were recorded, and  their validity was controlled by comparing the melting  curve characteristics of each PCR fragment produced with corresponding positive controls. For such  positive controls, which were in- cluded in each PCR run, DNA known to be methylated in the marker  regions was  used. A no template control using  water  as template was  also  included in each qMSP  run.  For evaluation, the  difference of the  Ct values of each marker with the  internal  control  ACHE was calculated. qMSPs  for ASTN1, DLX1, ITGA4, RXFP3 ans SOX17 yielding a difference between sample and internal control  ≤ 9.0 were scored pos- itive, for ZNF671 ≤10 was  scored positive. A GynTect assay was  scored positive,  if the  sum  of the  factors attributed to each marker  was 0.5 or higher (Table 1).

Results

For assessing methylation of the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, ZNF671 as well as the two internal marker  regions ACHE and IDS an intercalating dye-based qMSP assay was performed for each of the 105 patient samples  included in this  study.  The results obtained for the  60 samples from patients with a cytology  result  Pap  I were the basis for setting the  delta  Ct value limit ≤ 9 for all markers but ZNF671, for which the delta Ct value limit was set to ≤ 10.

To be scored valid, the Ct value for the control  marker  ACHE 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  obtained for the 45 CIN1+ samples.

All five carcinomas included in the study were scored GynTect-positive. Of the  26 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). When related to cytology findings, the following results were obtained for the CIN samples: of the  17 samples scored Pap  III or Pap  IIID in cytology, seven (= 41%) turned out to be GynTect-positive; of the 23 Pap IVa samples, 16 (= 69.6%) were GynTect-positive (Figure 1B).

Discussion

In previous studies  we  have  shown that  hypermethylation of CpG islands in proximity  to the  genes DLX1, ITGA4, RXFP3, SOX17, and ZNF671 correlated with the presence of precancerous cervical lesions and cervical cancer (Hansel et al., 2014).  The molecular diagnostic test GynTect based on these results allows the detection of these marker  regions in cervical smears collected in the denaturing specimen trans- port medium (STM), which  is originally used for QIAGEN’s DIGENE HPV test. Utilization of this  medium 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  media can  be used more flexibly. As a main  advantage, the  cellular 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.

In this study we evaluated whether our molecular diagnostic test GynTect is suitable for using  residual material from liquid-based cytology samples and such  fulfils this prerequisite. GynTect provides the possibility  to test if a woman who obtained an abnormal cytology finding in the Pap smear and/ or a positive HPV test result, has  a precancerous lesion  that requires follow-up and treatment. For this purpose we used samples for which the cytology  findings  and, for all Pap-abnormal  samples, the  histopathology results were  available for comparison. GynTect showed an excellent performance, since for all 105 samples valid test results could be obtained. The results obtained for the  two  internal  markers that  are tested with each patient sample demonstrate this.  In fact, the Ct values for these two internal  markers obtained for all 105  LBC samples are  much  lower  than  those 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  Ct values in relation to the controls was  set.  Using a delta  Ct threshold of 9 for the five markers ASTN1, DLX1, ITGA4, RXFP3, and SOX17 as well as a delta  Ct threshold of 10 for ZNF671, of the  60 samples with a normal cytology, Pap I, only one sample yielded a GynTect-positive result, implying that  the test has a very good specificity within this  group.  A larger  number of such  samples will definitely have to be examined to confirm  these results and this very high specificity of the test among healthy women.

As expected, all 5 cervical cancer samples included in the study were  detected by GynTect, and  that  by at least four GynTect markers. This very high sensitivity for cancer cas- es was already  shown previously (Hansel et al., 2014).  A detection rate  of >60% among the CIN3 samples examined also  confirms results obtained in previous studies (Hansel et al., 2014).  It is well-known  that  not  all CIN3 lesions proceed to cervical  cancer (McCredie  et  al., 2008),  although these  high-grade lesions  are   considered as precancer- ous  stages. In several observational studies CIN2/3  shortterm  regression rates around 30% were reported (Trimble et al., 2005,  2010,  2015; Grimm et al., 2012).  Very recently, Loopik et al. (2016)  have demonstrated in a retrospective study  that  in women < 25 years  the regression rate  of CIN2 lesions was as high as 71% (150  of 211  women followed after CIN2 diagnosis), and the overall progression rate in this study  was very low (15%).

The data  show that the GynTect score is related to the severity of the lesion  confirmed by histopathology. In fact, the higher the CIN grade, the more GynTect markers are positive in the  LBC 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  not taken  at the punctum maximum of the lesion. Altogether, the correlation between cytology finding and GynTect result  was even higher.

In conclusion, GynTect, a test which provides a triage  option for either HPV-based or cytology-based cervical  cancer screening, shows excellent results if performed on cervical scrape material in liquid-based cytology  media,  a prerequisite for employing such  a test in new screening programs.

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