Martial Guillaud

Real-time reflectance confocal microscopy: comparison of two-dimensional images and three-dimensional image stacks for detection of cervical precancer

Confocal microscopy can provide real-time, 2-D and 3-D images of the cellular morphology and tissue architecture features that pathologists use to detect precancerous lesions without the need for tissue removal, sectioning, and staining. The utility of 3-D confocal image stacks of epithelial tissue for detecting dysplasia has not yet been explored. We aim to extract morphometry and tissue architecture information from 2-D confocal reflectance images and 3-D image stacks from fresh, unstained cervical biopsies and compare their potential for detecting dysplasia. Nine biopsies are obtained from eight patients; confocal images are acquired pre- and postacetic acid at multiple epithelial depths in 1.5 mum-intervals. Postacetic acid images are processed to segment cell nuclei; after segmentation, 2-D images taken at 50 mum below the tissue surface, and the entire 3-D image stacks are processed to extract morphological and architectural features. Data are analyzed to determine which features gave the best separation between normal and high-grade cervical precancer. Most significant differences are obtained from parameters extracted from the 3-D image stacks. However, in all cases where the 2-D features were multiplicatively scaled by the depth of acquisition divided by the epithelial thickness or scaled by the scattering coefficient, the significance level is equal to or greater than the comparable feature extracted from the 3-D image stacks. A linear discriminant function previously developed to separate 19 samples of normal tissue and high-grade cervical precancer based on the nuclear-to-cytoplasm (N/C) ratio and epithelial scattering coefficient is prospectively applied to the nine biopsies examined to determine the accuracy with which it could separate normal tissue from cervical intra epithelial neoplasia (CIN) 23. For the entire data set of 28 biopsies, a sensitivity and specificity of 100% is produced using this discriminant function; the scattering coefficient provides more discriminative capacity than the N/C ratio. The success of the scaled 2-D image features has important implications for using confocal microscopy to detect precancer in the clinic. Acquisition of the epithelial thickness or scattering coefficient requires less time than 3-D image sets and little additional effort is required to gain the added information compared to 2-D images alone.

Human papillomavirus‐related cellular changes measured by cytometric analysis of DNA ploidy and chromatin texture

Image cytometry has provided two highly sensitive markers for the identification of the malignant potential of squamous lesions. Aneuploidy and chromatin texture have been investigated as quantitative measures of nuclear damage in premalignant lesions and carcinoma. Real‐time PCR methods have evolved to yield highly specific measurements of mRNA expression in very sparse cellular samples.

Automated Sputum Cytometry for Detection of Intraepithelial Neoplasias in the Lung

Background: Despite the benefits of early lung cancer detection, no effective strategy for early screening and treatment exists, partly due to a lack of effective surrogate biomarkers. Our novel sputum biomarker, the Combined Score (CS), uses automated image cytometric analysis of ploidy and nuclear morphology to detect subtle intraepithelial changes that often precede lung tumours. Methods: 2249 sputum samples from 1795 high-risk patients enrolled in ongoing chemoprevention trials were subjected to automated quantitative image cytometry after Feulgen-thionin staining. Samples from normal histopathology patients were compared against samples from carcinoma in situ (CIS) and cancer patients to train the CS. Results: CS correlates with several lung cancer risk factors, including histopathological grade, age, smoking status, and p53 and Ki67 immunostaining. At 50% specificity, CS detected 78% of all highest-risk subjects—those with CIS or worse plus those with moderate or severe dysplasia and abnormal nuclear morphology. Conclusion: CS is a powerful yet minimally invasive tool for rapid and inexpensive risk assessment for the presence of precancerous lung lesions, enabling enrichment of chemoprevention trials with highest-risk dysplasias. CS correlates with other biomarkers, so CS may find use as a surrogate biomarker for patient assessment and as an endpoint in chemoprevention clinical trials.

DNA Ploidy Cytometry Testing for Cervical Cancer Screening in China – Letter

We read the article of Tong et al. ([1][1]) with great interest. However, on closer analysis, we found several points that need clarification. Although the authors call this a “randomized controlled trial,” both tests were done on almost all of the study subjects and the data presented in Table

Targeting of chemoprevention to high-risk potentially malignant oral lesions: Challenges and opportunities

Worldwide, oral cancer is responsible for 170,000 deaths per year. Intervention to prevent this disease is a long sought after goal. Chemoprevention studies have focused on clinicopathological features of potentially malignant lesions (PML) in an effort to prevent their progression to cancer. However, prediction of future behavior for such lesions is difficult and remains a major challenge to such intervention. Different approaches to this problem have been tested in the past 20years. Early genetic progression models identified critical regions of allelic imbalance at 3p and 9p, and provided the basis for molecular markers to identify progressing PMLs. Subsequently, technological advances, such as genome-wide high-throughput array platforms, computer imaging, visualization technology and next generation sequencing, have broadened the scope for marker development and have the potential of further improving our ability to identify high-risk lesions in the near future either alone or in combination. In this article, we examine the milestones in the development of markers for PML progression. We emphasize the critical importance of networks among scientists, health professionals and community to facilitate the validation and application of putative markers into clinical practice. With a growing number of new agents to validate, it is necessary to coordinate the design and implementation of strategies for patient recruitment, integration of marker assessment, and the final translation of such approaches into clinical use.

Molecular fixative enables expression microarray analysis of microdissected clinical cervical specimens

Formalin-fixed tissue has been a mainstay of clinical pathology laboratories, but formalin alters many biomolecules, including nucleic acids and proteins. Meanwhile, frozen tissues contain better-preserved biomolecules, but tissue morphology is affected, limiting their diagnostic utility. Molecular fixatives promise to bridge this gap by simultaneously preserving morphology and biomolecules, enabling clinical diagnosis and molecular analyses on the same specimen. While previous reports have broadly evaluated the use of molecular fixative in various human tissues, we present here the first detailed assessment of the applicability of molecular fixative to both routine histopathological diagnosis and molecular analysis of cervical tissues. Ten specimens excised via the loop electrosurgical excision procedure, which removes conical tissue samples from the cervix, were cut into alternating pieces preserved in either formalin or molecular fixative. Cervical specimens preserved in molecular fixative were easily interpretable, despite featuring more eosinophilic cytoplasm and more recognizable chromatin texture than formalin-fixed specimens. Immunohistochemical staining patterns of p16 and Ki-67 were similar between fixatives, although Ki-67 staining was stronger in the molecular fixative specimens. The RNA of molecular fixative specimens from seven cases representing various dysplasia grades was assessed for utility in expression microarray analysis. Cluster analysis and scatter plots of duplicate samples suggest that data of sufficient quality can be obtained from as little as 50ng of RNA from molecular fixative samples. Taken together, our results show that molecular fixative may be a more versatile substitute for formalin, simultaneously preserving tissue morphology for clinical diagnosis and biomolecules for immunohistochemistry and gene expression analysis.

Fluorescence Visualization and Imaging: A Tool for Improving Patient Outcomes for Oral and (Hopefully) Other Cancers

Improved detection and delineation of pre-invasive and invasive cancers can be effected through recording the changes in optical properties detected by auto-fluorescence imaging, resulting in statistically improved patient outcomes (oral) and improved lesion detection (cervix).

Intermediate endpoint biomarkers for lung cancer chemoprevention

Given the demographics of current and ex-smoking populations in North America, lung cancer will be a major problem in the foreseeable future. Early detection and treatment of lung cancer holds great promise for the management of this disease. Unlike cervical cancer, the physical, complete removal/destruction of all dysplastic lesions in the bronchial tree is not possible; however, treatment of the lesions using a chemopreventive agent is. Intermediate biomarkers have been used to screen promising chemopreventive agents for larger population studies. We have examined the natural history of lung cancer development by following a group of subjects at high risk of developing lung cancer using fluorescence endoscopy to identify the areas of abnormality for biopsy. Approximately 900 biopsies have been collected in this fashion and graded by at least two experienced, expert pathologists. Using an interactive version of the Cyto-Savant (Oncometrics Imaging Corp.), cytometric and tissue architectural data were collected from these biopsies. Using only the data from the normal and invasive cancer biopsies, quantitative morphometric and architectural indices were generated and calculated for all the collected biopsies. These indices were compared with Loss of Heterozygosity (LOH) of ten sites commonly associated with cancer. These results and the application of these quantitative measures to two small chemoprevention studies will be reported.

Novel computational image analysis to predict regional nodal disease for early-stage oral cancer

Objective: Nodal disease (N+) for early-stage oral squamous cell carcinoma (OSCC) is the most significant prognostic factor for survival. There is a lack of effective predictor to justify prophylactic neck dissection. Quantitative tissue pathology (QTP) has shown its promise in providing an objective means for diagnosis and prognosis of many cancer types. We conducted a pilot study on the utilization of QTP to evaluate risk of nodal disease. Study design: Retrospective case-control study Subjects and methods: Histological sections from 15 primary tumors of clinically node-negative (N0) patients were stained with Feulgen-Thionin followed by acquisition of digital images and image processing to measure the mean and variance of nuclear phenotype and tissue architecture features from 45,253 nuclei of 45 tumor nests. Association between features and nodal disease outcome (N0 or N+) was investigated using nested analysis of variance adjusted by patient. Ability to discriminate between N0 and N+ was analyzed using multivariate logistic regression and receiver operating characteristics (ROC) curve analysis. P-value<0.05 (2-sided) was considered significant. Results: The N+ group presented higher mean values of chromatin condensation levels and cell density compared to those of the N0 group. ROC curve showed a strong discriminative ability of chromatin condensation levels between the N+ and N0 groups with a sensitivity and specificity of 1.0 and 0.75, respectively. Conclusion: This study reports the first-ever data on QTP as a risk assessment tool for nodal disease in early-stage OSCCs. Such computational imaging analysis potentially provides a new objective approach to predict regional nodal disease. Correspondence to: Dr. Catherine F. Poh, Department of Oral Medical and Biological Sciences, Faculty of Dentistry, The University of British Columbia, 2199 Wesbrook Mall, Vancouver BC, V6T 1Z3, Canada; E-mail: cpoh@dentistry.ubc.ca

Abstract A17: Using high‐throughput imaging cytometry to unmask the true nature of oral lesions in a high‐risk community

Vancouver9s Downtown Eastside is a high‐risk community for oral cancer. Previous study has shown its prevalence of tobacco and alcohol consumption and high incidence of oral cancer (one cancer identified in 150 residents screened). Additionally, oral infection and inflammation are commonly seen in this community. In British Columbia, we are evaluating several technologies and their interactions for the screening and early detection. Direct fluorescence visualization (FV) has shown its high sensitivity for the detection of oral cancer or precancer, but can also highlight other oral conditions which are commonly seen within community setting. We start to look at the utility of imaging cytometry as an adjunct tool in oral cancer screening. There is an urgent need to develop an effective strategy for oral cancer screening in such a high risk community. Objectives: 1) To using imaging cytometry to measure the altered nuclear phenotype (cNPS) and 2) To see if cNPS can increase the accuracy of using FV only for oral screening. Methods: From 2004/11–2009/2, we have collected 355 exfoliative cell samples using a small curved interdental tooth brush from this high‐risk community. Samples were spun down onto slides, the DNA quantitatively labeled and automatically scanned by the cyto‐savant®. For each object (nucleus/debris) imaged ∼110 features were calculated and used by a cell recognition decision tree to differentiate cells from debris. Results: Among 355 brushings, 4 from cancerous sites, 90 from non cancerous common oral lesions (60, trauma; 28, inflammation; 12, infection), and from tongue with no lesion under clinical white light and FV examinations. Using previously trained algorithm with 84% sensitivity and 97% specificity to examine these samples, there was no difference between gender, age groups, smoking habit, and immune status (presence of HIV infection). This algorithm can correctly identified 3 out of 4 high‐grade lesions and 89% normal cases. For those non‐cancerous common oral lesions, using FV followed with the examination of cNPS at the FV loss area can drastically increase the accuracy from 10% to 83% (trauma, 13% to 90%; inflammation, 6% to 78%; infection 0% to 75% respectively). Conclusion: The pilot results indicate support the potential usage of the combination of direct FV and imaging cytometry in oral cancer screening in a high‐risk community. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A17.