Burcu Sengüven

Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues

Aim: Discussing a protocol involving xylene-ethanol deparaffinization on slides followed by a kit-based extraction that allows for the extraction of high quality DNA from FFPE tissues. Methods: DNA was extracted from the FFPE tissues of 16 randomly selected blocks. Methods involving deparaffinization on slides or tubes, enzyme digestion overnight or for 72 hours and isolation using phenol chloroform method or a silica-based commercial kit were compared in terms of yields, concentrations and the amplifiability. Results: The highest yield of DNA was produced from the samples that were deparaffinized on slides, digested for 72 hours and isolated with a commercial kit. Samples isolated with the phenol-chloroform method produced DNA of lower purity than the samples that were purified with kit. The samples isolated with the commercial kit resulted in better PCR amplification. Conclusion: Silica-based commercial kits and deparaffinized on slides should be considered for DNA extraction from FFPE.

Histological comparison of healing following tooth extraction with ridge preservation using enamel matrix derivatives versus Bio- Oss Collagen: a pilot study

The goal of the present clinical study was to evaluate new bone formation in human extraction sockets augmented with enamel matrix derivatives (EMD) and Bio-Oss Collagen. Patients with symmetrical single-rooted teeth in the bilateral quadrants of the upper jaw condemned for extraction participated in this study. Following extraction, the sockets (20 sockets) were randomly augmented using either EMD or Bio-Oss Collagen. After 3 months of healing, bone biopsies were obtained and prepared for histological analyses. Dental implants were then placed. Implant stability quotient (ISQ) readings were obtained for each implant at the time of surgery and at 1 and 3 months postoperatively. The mean new bone formation was 34.57 ± 25.67% in the EMD sites and 28.80 ± 16.14% in the Bio-Oss Collagen sites. There was no significant difference between the groups. The ISQ values were significantly higher for the implants placed in the EMD sites at the first and third months, but no significant differences were observed in the ISQ values for the implants placed in the Bio-Oss Collagen sites. The augmentation of the extraction sockets with EMD or Bio-Oss Collagen leads to similar behaviour in bone regeneration.

Double-application of platelet-rich plasma on bone healing in rabbits

Objective: Platelet-rich plasma (PRP) is considered to enhance bone formation especially at early stages of wound healing, depending on the limited and short life-span of platelets and growth factors. The aim of this study was to evaluate efficacy of double-application of PRP (DA-PRP) on bone healing in a rabbit calvarial defect model. Study design: Twenty-eight rabbits, each had two surgically prepared calvarial bone defects (10mm diameter), were included in this study and randomly divided into six groups. Defects (n=56) were treated with single-application of PRP (SA-PRP)(n=10), SA-PRP and beta-tricalciumphosphate (SA-PRP+TCP)(n=10), DA-PRP (n=8), DA-PRP and beta-tricalciumphosphate (DA-PRP+TCP)(n=8), beta-tricalciumphosphate (TCP)(n=10) or left empty (Control)(n=10). Animals were sacrificed at 30 days postoperatively. Results: The new bone (NB%) and defect fill (DF%) percentages were calculated from histological slides by image-analyzer software and statistically analysed. All test groups showed higher NB% than control, but differences among all groups were insignificant. The TCP treated groups had significantly higher DF% than groups treated without TCP, however the DF% differences between control, SA-PRP and DA-PRP or TCP, SA-PRP+TCP or DA-PRP+TCP were insignificant. Conclusion: Although new bone formation was histomorphologically remarkable at double-application PRP groups, statistical analyses of the histomorphometric data revealed no significant difference. Key words: Platelet-Rich Plasma, double application, bone formation, wound healing.

Tetraspanins CD9 and CD151, epidermal growth factor receptor and cyclooxygenase-2 expression predict malignant progression in oral epithelial dysplasia.

Background:Prognostic biomarkers aim to improve on the current inadequate method of histological assessment to identify patients with oral epithelial dysplasia at greatest risk of malignant transformation. We aimed to assess the prognostic ability of six protein biomarkers linked to the epidermal growth factor receptor (EGFR) pathway, including three tetraspanins, in a large multicentre oral dysplasia cohort.Methods:One hundred and forty-eight cases with varying degrees of epithelial dysplasia underwent immunohistochemical assessment for CD9, CD151, CD82, EGFR, Her-2, and COX-2. Scoring was performed independently by two observers. Univariate analyses using both logistic and Cox regression models and a multivariate regression were performed.Results:Malignant progression was significantly greater in those cases with decreased expression of CD9 (P=0.02), and increased expression of CD151 (P=0.02), EGFR (P=0.04), and COX-2 (P=0.003). Histological grade (P=0.0002) and morphology (P=0.03) were also prognostic, whereas smoking and alcohol were not. The optimal combination by backward-variable selection was of histological grade (hazard ratio (HR) 1.64; 95% CI 1.12, 2.40), COX-2 overexpression (HR 1.12; 1.02, 1.24) and CD9 underexpression (HR 0.88; 0.80, 0.97). CD82 and Her-2 demonstrated no prognostic ability.Conclusion:This is the first study of the expression and prognostic potential of the tetraspanins in oral dysplasia. A combination of certain biomarkers with clinical factors appeared to improve the accuracy of determining the risk of malignancy in individuals with oral dysplasia. These findings may also offer potential new therapeutic approaches for this condition.

The clonal relationships between pre‐cancer and cancer revealed by ultra‐deep sequencing

The study of the relationships between pre‐cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre‐cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra‐deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre‐invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre‐cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre‐cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre‐cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588. Copyright

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low‐coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non‐metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

Effect of the Topical Use of the Antioxidant Taurine on the Two Basement Membrane Proteins of Regenerating Oral Gingival Epithelium

BACKGROUND The essential amino acid taurine has important physiologic and pathologic roles, and has been shown to have osmoregulatory, antioxidative, antiapoptotic, anti-inflammatory, and antilipid activities. However, the response of oral gingival epithelium to taurine during wound healing remains unclear. The goal of this study is to evaluate the expression of laminin 5 and Type IV collagen histologically in regenerating gingival epithelium after direct application of taurine on incised human gingival samples. METHODS The study was conducted on 16 gingival samples obtained from gingivectomy specimens of eight adult patients with generalized gingival overgrowth. The samples were divided into two groups: gingiva with 1% taurine-hydrated collagen membrane (n = 8) and saline-hydrated collagen membrane (n = 8) applied specimens. The length of the newly formed epithelium on the wound surface and inflammation was assessed on hematoxylin and eosin-stained sections. Basement membrane formation was evaluated by detection of laminin 5 and Type IV collagen expressions on immunohistochemically stained samples. RESULTS Complete new epithelial formation was observed in 1% taurine-treated gingivectomy specimens, whereas incomplete regeneration of the epithelium was observed in control gingivectomy specimens (P <0.05). The length of the newly formed epithelium showed a negative correlation with inflammation in the taurine group (P = -0.712; P <0.05). Immunoreactivity for both laminin 5 and Type IV collagen did not show any significant difference between groups. CONCLUSION The local application of taurine-hydrated collagen membrane on human gingival wounds demonstrated the histologic evidence of rapid reepithelization with taurine.

Investigation of p16INK4a as a prognostic biomarker in oral epithelial dysplasia

BACKGROUND Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. METHODS One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. RESULTS Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). CONCLUSION Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

The landscape of genetic alterations in ameloblastomas relates to clinical features

Ameloblastoma is a mostly benign, but locally invasive odontogenic tumor eliciting frequent relapses and significant morbidity. Recently, mutually exclusive mutations in BRAF and SMO were identified causing constitutive activation of MAPK and hedgehog signaling pathways. To explore further such clinically relevant genotype-phenotype correlations, we here comprehensively analyzed a large series of ameloblastomas (98 paraffin block of 76 patients) with respect to genomic alterations, clinical presentation, and histological features collected from the archives of three different pathology centers in France, Germany, and Turkey. In good agreement with previously published data, we observed BRAF mutations almost exclusively in mandibular tumors, SMO mutations predominantly in maxillary tumors, and single mutations in EGFR, KRAS, and NRAS. KRAS, NRAS, PIK3CA, PTEN, CDKN2A, FGFR, and CTNNB1 mutations co-occurred in the background of either BRAF or SMO mutations. Strikingly, multiple mutations were exclusively observed in European patients, in solid ameloblastomas and were associated with a very high risk for recurrence. In contrast, tumors with a single BRAF mutation revealed a lower risk for relapse. We here establish a comprehensive landscape of mutations in the MAPK and hedgehog signaling pathways relating to clinical features of ameloblastoma. Our data suggest that ameloblastomas harboring single BRAF mutations are excellent candidates for neo-adjuvant therapies with combined BRAF/MEK inhibitors and that the risk of recurrence maybe stratified based on the mutational spectrum.

Primary oral myeloid sarcoma: Report of a case

Myeloid sarcoma is defined as a tumor mass of immature myeloid cells that may be observed in a variety of locations including bone, skin, lymph nodes and soft tissues. However, oral involvement of myeloid sarcoma is extremely rare. These tumors are considered as specific lesions of acute myeloid leukemia. We present a case of a myeloid sarcoma of the upper vestibular gingiva in a 29-year-old woman who has no hematologic disease history. Multiple metastases were found in floor of the nasal cavity, left breast, and left lacrimal gland 12 months after primary diagnosis.