The role of methylation in metastasis of oral squamous cell carcinoma: Understanding the OSCC methylome

Background: Oral Squamous Cell Carcinoma (OSCC) is characterized by an increasing incidence and a 60% 5-year survival. The most important prognostic factor for OSCC is the presence of lymph node (LN) metastases but the detection of LN metastases in the clinic by palpation and imaging is inaccurate resulting in under and overtreatment of patients. To improve LN diagnosis new detection methods are needed. DNA methylation studies can be used to identify novel biomarkers, as epigenetics have been established as an important regulator of metastatic potential. The aim of this study is to identify new DNA methylation markers that predict LN metastasis in OSCC. Materials and Methods: Genome-wide methylation patterns of metastatic (n = 6) and non-metastatic OSCC (n = 6) were assessed using MethylCap- Seq. Subsequently, analysis was performed on the most differentially methylated loci to identify pathways and genomic loci associated with LN metastasis. Additionally, the methylation data were combined with expression data of 222 OSCC patients acquired by an expression microarray with a 696 gene panel associated with N-status in OSCC. Finally, we validated these findings on the OSCC samples (n = 174) in The Cancer Genome Atlas (TCGA). Results: We found that genes on chromosome 7 are the most hypermethylated in metastatic OSCC in comparison to non-metastatic OSCC, more specifically a four million bp loci around the EGFR gene. In total 26 genes were found to be differentially methylated by MethylCap- Seq as well as differentially expressed by microarray. In the OSCC TCGA validation cohort, all 26 genes are significantly differentially expressed between metastatic and non-metastatic OSCC. For these 26 differentially expressed genes 88 probes were found in the TCGA Infinium 450k methylation data that are both significantly differentially methylated between the two groups and correlated with mRNA levels. Finally, six of these probes overlapped with the genomic regions annotated by MethylCap-Seq, representing five genes. Conclusion: We identified chromosomal loci, pathways and gene promoters associated with LN metastasis in OSCC patients. For 10 genes the differential methylation, expression and correlation between methylation and expression was validated on an independent OSCC cohort from the TCGA database. Increased understanding of the metastatic OSCC methylome will contribute to the identification of DNA methylation markers, pathways and potential therapeutic targets in OSCC primary tumors that will improve chances of providing the proper lymph node treatment and may result in improvement of survival and quality of life

Prognostic cell biological markers in cervical cancer patients primarily treated with (chemo)radiation: a systematic review

The aim of this study was to systematically review the prognostic and predictive significance of cell biological markers in cervical cancer patients primarily treated with (chemo)radiation. A PubMed, Embase, and Cochrane literature search was performed. Studies describing a relation between a cell biological marker and survival in >/=50 cervical cancer patients primarily treated with (chemo)radiation were selected. Study quality was assessed, and studies with a quality score of 4 or lower were excluded. Cell biological markers were clustered on biological function, and the prognostic and predictive significance of these markers was described. In total, 42 studies concerning 82 cell biological markers were included in this systematic review. In addition to cyclooxygenase-2 (COX-2) and serum squamous cell carcinoma antigen (SCC-ag) levels, markers associated with poor prognosis were involved in epidermal growth factor receptor (EGFR) signaling (EGFR and C-erbB-2) and in angiogenesis and hypoxia (carbonic anhydrase 9 and hypoxia-inducible factor-1alpha). Epidermal growth factor receptor and C-erbB-2 were also associated with poor response to (chemo)radiation. In conclusion, EGFR signaling is associated with poor prognosis and response to therapy in cervical cancer patients primarily treated with (chemo)radiation, whereas markers involved in angiogenesis and hypoxia, COX-2, and serum SCC-ag levels are associated with a poor prognosis. Therefore, targeting these pathways in combination with chemoradiation may improve survival in advanced-stage cervical cancer patients

CD103+ tumor-infiltrating lymphocytes are tumor-reactive intraepithelial CD8+ T cells associated with prognostic benefit and therapy response in cervical cancer

Contains fulltext : 178711.pdf (Publisher’s version ) (Open Access)Human papilloma virus (HPV)-induced cervical cancer constitutively expresses viral E6/E7 oncoproteins and is an excellent target for T cell-based immunotherapy. However, not all tumor-infiltrating T cells confer equal benefit to patients, with epithelial T cells being superior to stromal T cells. To assess whether the epithelial T cell biomarker CD103 could specifically discriminate the beneficial antitumor T cells, association of CD103 with clinicopathological variables and outcome was analyzed in the TCGA cervical cancer data set (n = 304) and by immunohistochemistry (IHC) in an independent cohort (n = 460). Localization of CD103+ cells in the tumor was assessed by immunofluorescence. Furthermore, use of CD103 as a response biomarker was assessed in an in vivo E6/E7+ tumor model. Our results show that CD103 gene expression was strongly correlated with cytotoxic T cell markers (e.g. CD8/GZMB/PD1) in the TCGA series. In line with this, CD103+ cells in the IHC series co-expressed CD8 and were preferentially located in cervical tumor epithelium. High CD103+ cell infiltration was strongly associated with an improved prognosis in both series, and appeared to be a better predictor of outcome than CD8. Interestingly, the prognostic benefit of CD103 in both series seemed limited to patients receiving radiotherapy. In a preclinical mouse model, HPV E6/E7-targeted therapeutic vaccination in combination with radiotherapy increased the intratumoral number of CD103+ CD8+ T cells, providing a potential mechanistic basis for our results. In conclusion, CD103 is a promising marker for rapid assessment of tumor-reactive T cell infiltration of cervical cancers and a promising response biomarker for E6/E7-targeted immunotherapy

The epidermal growth factor receptor pathway in relation to pelvic lymph node metastasis and survival in early-stage cervical cancer

The objective of this study is to correlate the expression of epidermal growth factor receptor (EGFR) components with clinical behavior of early-stage cervical cancer. Tissue samples of 336 consecutive Federation of International Gynecologists and Obstetricians stage IB-IIA cervical cancer patients all treated primarily by radical surgery were collected. Clinicopathologic and follow-up data were prospectively obtained during standard treatment and follow-up. As representatives for the EGFR pathway, expression of EGFR, pEGFR, PTEN, pAKT, and pERK was assessed by immunohistochemistry on tissue microarrays. Positive immunostaining was observed for EGFR in 32.1%, for pEGFR in 21.0%, for PTEN in 38.3%, for pAKT in 5.3%, and for pERK in 4.3% of tumor samples. Positive EGFR immunostaining was associated with squamous cell carcinoma of the cervix (odds ratio [OR], 7.41; 95% confidence interval [CI], 3.38-16.23, P < .001), negative pEGFR immunostaining with poor differentiation (OR, 0.39; 95% CI, 0.20-0.73, P = .004), and negative PTEN immunostaining with metastatic pelvic lymph nodes (OR, 0.51; 95% CI, 0.30-0.90, P = .019). In multivariate analysis, only pelvic lymph node metastasis (hazard ratio, 6.11; 95% CI, 3.46-10.77, P < .001) and poor differentiation (hazard ratio, 1.91; 95% CI, 1.12-3.26, P = .018) were related to disease-specific survival. In early-stage cervical cancer, loss of PTEN expression is associated with pelvic lymph node metastasis, suggesting PTEN to be one of the tumor suppressor genes affecting pelvic lymph node metastasis. However, expression of EGFR pathway components does not appear to have prognostic impact in surgically treated early-stage cervical cancer

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

Contains fulltext : 137734.pdf (publisher's version ) (Closed access)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 (kappa=0.75, P<0.001) and 90% for C13ORF18 (kappa=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

A four gene methylation marker panel as triage test in hr-HPV 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 (hr)-HPV positive patients. First, high through-put 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(R) 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 4-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 4-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 4-gene methylation panel might provide an alternative triage test after primary hr-HPV testing

The role of ATM and 53BP1 as predictive markers in cervical cancer

Treatment of advanced-stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the 'DNA damage response' (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in patients with cervical cancer treated with (chemo)radiation. In a panel of cervical cancer cell lines, inactivation of ataxia telangiectasia mutated (ATM) or its substrate p53-binding protein-1 (53BP1) clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radiosensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance. To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of patients with advanced-stage cervical cancer (n = 375) treated with (chemo)radiation. High levels of phosphorylated (p-)ATM [p = 0.006, hazard ratio (HR) = 1.817] were related to poor locoregional disease-free survival. Furthermore, high levels of p-ATM predicted shorter disease-specific survival (p = 0.038, HR = 1.418). The presence of phosphorylated 53BP1 was associated with p-ATM (p = 0.001, odds ratio = 2.206) but was not related to any clinicopathological features or survival. In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve the efficacy of (chemo)radiation