Sequence homologies between the HPV16 genome and the two adjoining genes <i>TP63</i> and <i>LEPREL1</i>.

<p>Three homologous stretches of up to 50 nucleotides are shown. The number of exact nucleotide matches is given in brackets (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039632#pone.0039632.s001" target="_blank">Sequences S1</a>). The DNA loop between the two homologies located on <i>LEPREL1</i> comprises 102.689 nucleotides; the second loop 269.968 nucleotides. Grey dots refer to the approximate location of the corresponding viral-cellular fusion transcripts detected in tumors D3829, T2107 and T4335 (from left to right).</p

Summary of all viral-cellular fusion transcripts analysed.

1<p>Common and rare fragile sites located at a distance of up to 5 Mb adjacent to the integration locus. Rare fragile sites are shown in italics.</p>‡<p>n.a.: not applicable, because fusion transcript is in antisense orientation.</p

Genes affected by HPV integration at least twice in individual tumors.

‡<p>n.s.: not specified; n.a.: not applicable, because fusion transcript is in antisense orientation; ^†integration sites without reference result from this work.</p>1<p>Two fusion transcripts were found in T2107; ²refers to sequenced integration sites; ³TP73L alias TP63; ⁴<i>sweeker</i> in Kraus 2008 is no longer listed in any database; ⁵VMP1 alias TMEM49; ⁶DKFZP566I133 alias TMEM49.</p

Chromosomal hotspots for HPV integration.

<p>Depicted are integration sites located within the cytogenetic bands 3q28 (A), 4q13.3 (B), 8q24.21 (C), 13q22.1 (D) and 17q21.2 (E). Light blue arrows: genes affected by HPV integration; red arrows: HPV fusion transcripts described in this work; grey arrows: HPV fusion transcripts described by Kraus et al. 2008; green arrows: fragile site; dark blue arrows: microRNAs.</p

Diagnostic performance of the DNA methylation marker panel for hrHPV-positive cervical scrapes in relation to histopathology (sampling 3).

<p>For HPV-genotyping see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091905#pone.0091905.s006" target="_blank">Table S4</a>.</p

Flowchart showing all steps performed for marker identification, verification and validation.

<p>SCC: squamous cell carcinoma; CIN: cervical intraepithelial neoplasia; CxCa: cervical carcinoma; MSP: methylation specific PCR; hrHPV: high-risk HPV.</p

DNA methylation analysis using bisulfite-treated DNA from cervical scrapes of 217 women with histologically confirmed cervical disease status (sampling 3).

<p>Total number and percentage, with 95% confidence intervals, of samples which were methylation-positive for at least two (upper part of table) or three (lower part of table) of five DNA marker regions.</p

Diagnostic performance of the methylation marker panel (sampling 3) with (a) and without (b) cancer cases.

<p>To be scored methylation positive if at least 2 of 5 markers were methylated. P-values refer to Fisher exact test, comparing test performance by age-group.</p

Projection of methylation test performance (scored as test-positive if at least 2 of 5 markers were methylated) in hrHPV positive women ≥30 years of age originated from a screening population [20].

<p><i>sensitivity = Σ (p_d*m_d)/Σ p_d specificity = Σ (p_n*(1-m_n))/Σ p_n.</i></p><p>p - proportion of women in the target population, m - proportion of methylation-positive women per group, d - group indices diseased, n - group indices non-diseased, NPV - negative predictive value, PPV - positive predictive value, prev – prevalence.</p

Single-marker qMSP experiments with bisulfite-treated DNA isolated from cervical scrapes (sampling 2) of hrHPV-negative women (n = 76), hrHPV-positive women with normal colposcopy (n = 91), and hrHPV-positive women with histologically confirmed CIN3 (n = 45) and cervical cancer (CxCa; n = 50).

<p>By using the algorithm “at least 2 of 5 markers” need to be methylated in order to score the sample methylation positive one of 8 adenocarcinoma and one of 42 SCC were false negative.</p