Torsten Wilhelm Remmerbach

Cytologic and DNA-cytometric early diagnosis of oral cancer

Objective. The aim of this prospective study was to report on the diagnostic accuracy of conventional oral exfoliative cytology taken from white‐spotted, ulcerated or other suspicious oral lesions in our clinic. In addition we checked DNA‐image cytometry as an adjuvant diagnostic tool. Our hypothesis is that DNA‐aneuploidy is a sensitive and specific marker for the early identification of tumor cells in oral brushings. Study design. 251 cytological diagnoses obtained from exfoliative smears of 181 patients from macroscopically suspicious lesions of the oral mucosa and from clinically seemingly benign oral lesions which were exisiced for establishing histological diagnoses were compared with histological and/or clinical follow‐ups of the respective patients. Additionally nuclear DNA‐contents were measured after Feulgen restaining using a TV image analysis system. Results. Sensitivity of our cytological diagnosis on oral smears for the detection of cancer cells was 94.6%, specificity 99.5%, positive predictive value 98.1% and negative predictive value 98.5%. DNA‐aneuploidy was assumed if abnormal DNA‐stemlines or cells with DNA‐content greater 9c were observed. On this basis the prevalence of DNA‐aneuploidy in smears of oral squamous cell carcinomas in situ or invasive carcinomas was 96.4%. Sensitivity of DNA‐aneuploidy in oral smears for the detection of cancer cells was 96.4%, specificity 100%, positive predictive value 100% and negative 99.0%. The combination of both techniques increased the sensivity to 98.2%, specificity to 100%, positive predictive value to 100% and negative to 99.5%. Conclusions. Brush cytology of all visible oral lesions, if they are clinically considered as suspicious for cancer, are an easily practicable, cheap, non‐invasive, painless, safe and accurate screening method for detection of oral precancerous lesions, carcinoma in situ or invasive squamous cell carcinoma in all stages. We conclude that DNA‐image cytometry is a very sensitive, highly specific and objective adjuvant tool for the early identification of neoplastic epithelial cells in oral smears.

Diagnostic value of nucleolar organizer regions (AgNORs) in brush biopsies of suspicious lesions of the oral cavity

Objective: The aim of this retrospective study was to report on the diagnostic accuracy of AgNOR‐analysis as an adjunctive diagnostic tool of conventional oral exfoliative cytology taken from suspicious lesions in our clinic. Study design: Cytological diagnoses obtained from brush biopsies of macroscopically suspicious lesions of the oral mucosa from 75 patients (final diagnoses: 53 histologically proven squamous cell carcinomas, 11 leukoplakias and other inflammatory oral lesions) and from 11 patients with normal mucosa as a negative control group were compared with histological and/or clinical follow‐ups. Five smears were doubtful and seven suspicious for tumor cells in the cytologic report. Number of AgNORs were counted in 100 squamous epithelial cell‐nuclei per slide after silver‐restaining. Results: Sensitivity of our cytological diagnosis alone on oral smears for the detection of squamous carcinomas was 92.5%, specificity 100%, positive predictive value was 100% and negative 84.6%. The best cut‐off value of the mean number of AgNOR dots per nucleus distinguishing benign from malignant cells was 4.8. The percentage of nuclei with more than three AgNORs had a cut‐off level of 70%. Applying these methods to twelve doubtful or suspicious cytological diagnoses we were able to correctly establish the diagnosis of malignancy in ten cases of histologically proven cancers and to reveal benignity in two histologically proven cases. Thus we achieved a positive and negative predictive value of 100% each. Conclusions: Smears from brushings of visible oral lesions, if clinically considered as suspicious for cancer, are an easily practicable, non‐invasive, painless, safe and accurate screening method for detection of oral cancerous lesions. We conclude that AgNOR‐analysis may be a useful adjunct to other methods in routine cytological diagnosis of oral cancer that can help to solve cytologically suspicious or doubtful cases. Colour figures can be viewed on http://www.esacp.org/acp/2003/25‐3/remmerbach.htm.

Earliest Detection of Oral Cancer Using Non-Invasive Brush Biopsy Including DNA-Image-Cytometry: Report on Four Cases

Objective: We describe four patients presenting early oral cancers, detected cytologically on non‐invasive brush biopsies including DNA‐image cytometry as an adjunctive method before histology on scalpel biopsies confirmed the evidence of malignancy. Methods: Brush biopsies were performed and smears thereof investigated cytologically. After Feulgen restaining, DNA‐measurements were performed using a DNA‐Image‐Cytometer. Case reports: Oral squamous cell carcinomas were diagnosed cytologically in macroscopically suspicious lesions and malignancy confirmed by DNA‐cytometry. The initially performed scalpel biopsies did neither supply evidence of oral cancer nor of severe dysplasia. After at least one to 15 months the occurrence of cancer was finally proven histologically on a second scalpel biopsy each (three microinvasive and one in situ carcinoma). Conclusion: Non‐invasive brush biopsies are a suitable instrument for early cytologic detection of cancer of the mouth. DNA‐image‐cytometry, as an adjunctive method, can be used to confirm the cytologic diagnosis or suspicion of cancer in patients with doubtful lesions (dysplasias). DNA‐aneuploidy is a marker for (prospective) malignancy in smears of the oral cavity, which may detect malignancy months prior to histology. In future this method could be used as a mass screening tool in dentists practise. Colour figures can be viewed on http://www.esacp.org/acp/2003/25‐4/remmerbach.htm.

Principles of oral surgery for prevention of bisphosphonate-related osteonecrosis of the jaw

OBJECTIVES Principles and workflow are described to prevent bisphosphonate-related osteonecrosis of the jaw (BRONJ) when oral surgery is necessary in patients taking bisphosphonates. MATERIAL AND METHOD A total of 117 surgical procedures were performed on 68 patients taking bisphosphonates. The basis of the treatment was (1) use of perioperative antibiotics; (2) after dentoalveolar surgical procedures, bone edges were smoothed and mucoperiosteal flaps were prepared to ensure tension-free wound closure; (3) sutures were not removed until 14 days postsurgery; (4) long-term results were evaluated. RESULTS Ninety-seven percent of all procedures were simple and showed no complications. Only 3 patients with a long history of intravenous bisphosphonate medication developed BRONJ within 4 weeks after surgery. No patient receiving oral bisphosphonates developed BRONJ. No long-term failure was observed when primary wound healing was successful. CONCLUSIONS The high success rate of the described surgical procedures imply dentoalveolar surgery with low risk of development of BRONJ is possible for patients taking bisphosphonates.

Organogallium(III) complexes as apoptosis promoting anticancer agents for head and neck squamous cell carcinoma (HNSCC) cell lines.

Organogallium(III) dinuclear (1-9) and tetranuclear (10) complexes present potential therapeutic agents for the treatment of various types of cancer. The antiproliferative activity of 1-10 was evaluated with cell lines of head and neck squamous cell carcinomas, e.g. HN (soft palate), Cal27, Cal33 (tongue) and FaDu (hypopharynx) cell lines. The activity of compound 8 is comparable with that of cisplatin on cell line Cal27 (IC(50) 4.6 μM for both compounds). The mode of cell death induced, caspase activity and cell cycle analysis were evaluated for potential hit compounds 3, 5 and 8 Potential hit compounds 3, 5 and 8 were further evaluated for the mode of cell death, caspase activity and cell cycle analysis. Apoptosis induced by compounds 3, 5 and 8 on Cal27 and FaDu cells was confirmed by DNA laddering , as well as acridine orange (AO) and ethidium bromide (EB) double staining. These compounds (3, 5 and 8) induced caspase-independent apoptosis (within 4 h of action) in cell line Cal27. Extrinsic-mediated apoptosis associated with caspase 8 and 3 activation is the main mode of cytotoxicity induced on FaDu cells by compounds 3, 5 and 8. Cell cycle perturbations caused by these compounds are also observed. Our data suggest that compounds 3, 5 and 8 should be studied further for the treatment of head and neck cancer.

Evaluation of single-cell biomechanics as potential marker for oral squamous cell carcinomas: a pilot study.

OBJECTIVES Early detection of oral cancer is a major health issue. The objective of this pilot study was to analyze the deformability of healthy and cancer cells using a microfluidic optical stretcher (OS). MATERIAL AND METHODS Different cancer cell lines, primary oral cancer cells, and their healthy counterparts were cultivated and characterized, respectively. A measurable deformation of the cells along the optical axis was detected, caused by surface stress, which is optically induced by the laser power. RESULTS All cells revealed a viscoelastic extension behavior and showed a characteristic deformation response under laser light exposure. The CAL-27/-33 cells exhibited the highest relative deformation. All other cells achieved similar values, but on a lower level. The cytoskeleton reacts sensitively of changing environmental conditions, which may be influenced by growth behavior of the cancer specimens. Nevertheless, the statistical analysis showed significant differences between healthy and cancer cells. CONCLUSION Generally, malignant and benign cells showed significantly different mechanical behavior. Cancer-related changes influence the composition of the cytoskeleton and thus affect the deformability, but this effect may be superimposed by cell cultivation conditions or cell doubling time. These influences had to be substituted by brush biopsies to minimize confounders in pursuing investigations.

Oral brush biopsy analysis by MALDI-ToF Mass Spectrometry for early cancer diagnosis

OBJECTIVES Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass Spectrometry holds promise as a non-invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may significantly improve the early diagnosis of oral cancer. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS. MATERIALS AND METHODS In the first step, a database consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as the basis for further classification of an additional 26 blinded samples including HNSCC, oral lesions and healthy mucosa. RESULTS By analysing spectral patterns of the blinded samples, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%. CONCLUSION ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to more accurate early diagnosis of HNSCC.

Oral brush biopsy analysis by matrix assisted laser desorption/ionisation-time of flight mass spectrometry profiling – A pilot study

Oral squamous cell carcinomas (OSCCs) often present as advanced tumours requiring aggressive local and regional therapy and result in significant functional impairment. The objective is to develop pre-symptomatic screening detection of OSCC by a brush biopsy method which is less invasive than the conventional biopsy for histology. Given the molecular heterogeneity of oral cancer, it is unlikely that even a panel of tumour markers would provide accurate diagnosis. Therefore, approaches such as the matrix-assisted-laser-desorption/ionisation-time-of-flight-mass-spectrometry (MALDI-TOF-MS) allow several biomarkers or peptide profile patterns to be simultaneously assessed. Brush biopsies from 27 patients with histology-proven OSCCs plus 40 biopsies from 10 healthy controls were collected. MALDI-TOF-MS profiling was performed and additional statistical analysis of the data was used to classify the disease status according to the biological behaviour of the lesion. For classification a support vector machine algorithm was trained using spectra of brush biopsy samples to distinguish healthy control patients from patients with histology-proven OSCC. MALDI-TOF-MS was able to distinguish between healthy patients and OSCC patients with a sensitivity of 100% and specificity of 93%. In summary, MALDI-TOF-MS in combination with sophisticated bioinformatic methods can distinguish OSCC patients from non-cancer controls with excellent sensitivity and specificity. Further improvement and validation of this approach is necessary to determine its feasibility to assist the pre-symptomatic detection of head and neck cancer screening in routine daily practice.

Liquid-based versus conventional cytology of oral brush biopsies: a split-sample pilot study

ObjectiveThe aim of this prospective split sample study was to evaluate the applicability of liquid-based cytology (LBC) of oral brush biopsies for detection of oral cancer.MethodsTwo different preparation techniques were investigated: the conventional transfer procedure to glass slides and the LBC preparation method. The obtainments of epithelial cells were performed five times with a nylon brush and transferred onto five glass slides. Additionally, the brushes, which were normally discarded, were stored in a fixative solution. Conventional slides and respective thin layers from a total of 113 cases were reviewed with both techniques.ResultsThin layers showed excellent morphology on a clear background, which allowed an accurate diagnosis. In contrast, the conventional glass slides showed significantly more blood contamination and cell overlapping. The sensitivity of conventional cytological diagnosis was 96.3%, the specificity archived 90.6%, the positive predictive value was 96.3% and the negative predictive value scored 90.6%. The sensitivity of the cytological diagnosis using thin layers archived 97.5%, the specificity was 68.8%, the positive predictive value revealed 88.76% and negative predictive value was 91.7%.ConclusionOur findings indicate that in oral cytology, LBC may replace other types of wet-fixed preparations using the full amount of collected cells, resulting in enhanced specimen quality archiving comparable values of diagnostic accuracy.Clinical relevanceLBC facilitates the cell collection due to simpler handling and less transfer errors by dentists and may improve the overall diagnostic accuracy of oral brush biopsies in future.

Phenotypic Heterogeneity of Streptococcus mutans in Dentin

Information concerning phenotypic heterogeneity of Streptococcus mutans in carious dentin is sparse. Matrix-assisted laser-desorption/ionization-time-of-flight mass-spectrometry (MALDI-TOF-MS) facilitates the phenotypic differentiation of bacteria to the subspecies level. To verify a supposed influence of restorative treatment on the phenotypic heterogeneity of S. mutans, we isolated and compared a total of 222 S. mutans strains from dentin samples of 21 human deciduous molars during caries excavation (T1) and 8 wks (T2) after removal of the temporary restoration. Phenotypic heterogeneity was determined by MALDI-TOF-MS and hierarchical clustering. Thirty-six distinct S. mutans phenotypes could be identified. Although indistinguishable phenotypes were found in the same teeth at T1 and T2, as well as in different teeth of individual participants, the phenotypic heterogeneity increased significantly, from 1.4 phenotypes per S. mutans-positive dentin sample at T1 to 2.2 phenotypes at T2. We attribute this to an adaptation of S. mutans to the modified environment under the restoration following caries excavation.