Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme.

BACKGROUND: The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology. METHODS: Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (< CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel. RESULTS: QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between < CIN2 and CIN3+ for all markers (p < 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p < 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation> 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified. CONCLUSION: The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients.

CD103-positive tissue resident memory-like CD8+ T cells (CD8CD103 TRM) are associated with improved prognosis across malignancies, including high-grade serous ovarian cancer (HGSOC). However, whether quantification of CD8, CD103 or both is required to improve existing survival prediction and whether all HGSOC patients or only specific subgroups of patients benefit from infiltration, remains unclear. To address this question, we applied image-based quantification of CD8 and CD103 multiplex immunohistochemistry in the intratumoral and stromal compartments of 268 advanced-stage HGSOC patients from two independent clinical institutions. Infiltration of CD8CD103 immune cell subsets was independent of clinicopathological factors. Our results suggest CD8CD103 TRM quantification as a superior method for prognostication compared to single CD8 or CD103 quantification. A survival benefit of CD8CD103 TRM was observed only in patients treated with primary cytoreductive surgery. Moreover, survival benefit in this group was limited to patients with no macroscopic tumor lesions after surgery. This approach provides novel insights into prognostic stratification of HGSOC patients and may contribute to personalized treatment strategies in the future.

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis.

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.

Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3.

BACKGROUND: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. RESULTS: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p ≤ 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. CONCLUSIONS: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts.

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma.

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.

DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women.

BACKGROUND: Primary high-risk human papillomavirus (hrHPV) testing in cervical cancer screening shows relatively low specificity, which makes triage testing necessary. In this study, DNA methylation analysis was compared with cytology for triage testing in hrHPV-positive women. Moreover, feasibility of DNA methylation analysis directly on brush-based self-sampled specimens was assessed. METHODS: Non-responding women from population-based screening were invited to self-collect a cervico-vaginal specimen for hrHPV testing; hrHPV-positive women were referred to a physician for triage liquid-based cytology. DNA methylation analysis was performed on 128 hrHPV-positive physician-collected triage samples and 50 matched brush self-samples with QMSP for C13ORF18, EPB41L3, JAM3 and TERT. RESULTS: In physician-taken triage material, DNA methylation analysis of JAM3 showed the highest combined specificity (88%) and sensitivity (82%) for detection of CIN3+, whereas cytology showed a specificity of 48% and a sensitivity of 91%. Out of 39 women with abnormal cytology and normal histology (false-positive by cytology), 87% were negative for JAM3 and 90% for C13ORF18 methylation. Agreement between DNA methylation analysis performed directly on the matched self-sampled material and physician-taken samples was 88% for JAM3 (κ=0.75, P<0.001) and 90% for C13ORF18 (κ=0.77; P<0.001). CONCLUSIONS: DNA methylation analysis as a triage test in hrHPV-positive women is an attractive alternative to cytology. Furthermore, DNA methylation is feasible directly on brush-based self-samplers and showed good correlation with matched physician-taken samples. Direct molecular triage on self-collected specimens could optimise the screening program, especially for non-responders, as this would eliminate the need for an additional physician-taken scraping for triage testing.

Checkpoint kinase 2 (Chk2) supports sensitivity to platinum-based treatment in high grade serous ovarian cancer.

OBJECTIVE: Platinum-based chemotherapy is the standard treatment in advanced stage high grade serous ovarian cancer (HGSOC), but the majority of patients will relapse with drug-resistant disease. Platinum induces double-strand DNA breaks and subsequently activation of the DNA damage response (DDR). Drugs targeting DDR pathway components have gained major interest to be combined with chemotherapy as they could increase the therapeutic window. In the present study, we investigated the activation status of the Ataxia Telangiectasia Mutated (ATM) signaling axis within the DDR in a large, well-defined cohort of advanced stage HGSOC patients. METHODS: Pre-therapy activation status of the ATM signaling axis of the DDR was determined by immunohistochemistry in 125 chemo-naive advanced stage HGSOC patients. Ovarian cancer cell lines with stable checkpoint kinase 2 (Chk2) knock down were used to study cell cycle distribution and survival in long-term clonogenic survival assays. RESULTS: All ATM signaling axis components showed high expression levels. In two well-defined groups with the largest contrast in treatment response, high expression of Chk2 was related to good response (OR=0.132; P=0.014). Chk2 depletion abrogated the cisplatin-induced S-phase cell cycle arrest and caused increased resistance to cisplatin in long-term clonogenic survival assays. CONCLUSIONS: Chk2 is related to good response to platinum-based chemotherapy in advanced stage HGSOC patients. Chk2-depleted ovarian cancer cell lines have diminished platinum sensitivity, suggesting that Chk2 should not be considered a therapeutic target along with platinum-based treatment in HGSOC patients.

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.

A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients.

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.

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 Cohen's 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.

The ErbB signalling pathway: protein expression and prognostic value in epithelial ovarian cancer.

Ovarian cancer is the most frequent cause of death from gynaecological cancer in the Western world. Current prognostic factors do not allow reliable prediction of response to chemotherapy and survival for individual ovarian cancer patients. Epidermal growth factor receptor (EGFR) and HER-2/neu are frequently expressed in ovarian cancer but their prognostic value remains unclear. In this study, we investigated the expression and prognostic value of EGFR, EGFR variant III (EGFRvIII), HER-2/neu and important downstream signalling components in a large series of epithelial ovarian cancer patients. Immunohistochemical staining of EGFR, pEGFR, EGFRvIII, Her-2/neu, PTEN (phosphatase and tensin homologue deleted on chromosome 10), total and phosphorylated AKT (pAKT) and phosphorylated ERK (pERK) was performed in 232 primary tumours using the tissue microarray platform and related to clinicopathological characteristics and survival. In addition, EGFRvIII expression was determined in 45 tumours by RT-PCR. Our results show that negative PTEN immunostaining was associated with stage I/II disease (P=0.006), non-serous tumour type (P=0.042) and in multivariate analysis with a longer progression-free survival (P=0.015). Negative PTEN staining also predicted improved progression-free survival in patients with grade III or undifferentiated serous carcinomas (P=0.011). Positive pAKT staining was associated with advanced-stage disease (P=0.006). Other proteins were expressed only at low levels, and were not associated with any clinicopathological parameter or survival. None of the tumours were positive for EGFRvIII. In conclusion, our results indicate that tumours showing negative PTEN staining could represent a subgroup of ovarian carcinomas with a relatively favourable prognosis.

Detection of telomerase, its components, and human papillomavirus in cervical scrapings as a tool for triage in women with cervical dysplasia.

AIM: To examine whether the detection of either telomerase and its components or high risk human papillomavirus (HPV) are of value in predicting the presence of cervical intraepithelial neoplasia (CIN) grade II/III in women referred because of cervical cytology reports showing at most moderate dyskaryosis. METHODS: Cervical scrapings of 50 women referred with cytological borderline, mild, or moderate dyskaryosis were analysed. Telomerase activity was assessed by a commercially available telomere repeat amplification protocol assay and its components human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) were assessed by reverse transcriptase polymerase chain reaction (PCR). HPV was detected by GP5+/6+ PCR enzyme immunosassay. Histological findings on colposcopy guided biopsies or excised cervical tissue were regarded as the final pathological diagnosis. The sensitivity and specificity for detecting CIN II/III were calculated. RESULTS: Twenty eight women were diagnosed with CIN II/III. Telomerase activity was detected in none, hTR in 88%, hTERT in 23%, and high risk HPV was detected in 79% of these women. As a diagnostic test none of the described analyses combined a sensitivity of at least 90% with a specificity >or= 90%. Despite the small numbers, calculation of the 95% confidence intervals excluded a combined sensitivity and specificity of at least 90% for all of the evaluated parameters. CONCLUSIONS: Neither detection of telomerase or its components, nor detection of high risk HPV seem suitable for the triage of women with borderline, mild, and moderate cytological dyskaryosis.