EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple 'omics' approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.

Ex vivo confocal imaging with contrast agents for the detection of oral potentially malignant lesions.

OBJECTIVES: We investigated the potential use of real-time confocal microscopy in the non-invasive detection of occult oral potentially malignant lesions. Our objectives were to select the best fluorescence contrast agent for cellular morphology enhancement, to build an atlas of confocal microscopic images of normal human oral mucosa, and to determine the accuracy of confocal microscopy to recognize oral high-grade dysplasia lesions on live human tissue. MATERIALS AND METHODS: Five clinically used fluorescent contrast agents were tested in vitro on cultured human cells and validated ex vivo on human oral mucosa. Images acquired ex vivo from normal and diseased human oral biopsies with bench-top fluorescent confocal microscope were compared to conventional histology. Image analyzer software was used as an adjunct tool to objectively compare high-grade dysplasia versus low-grade dysplasia and normal epithelium. RESULTS: Acriflavine Hydrochloride provided the best cellular contrast by preferentially staining the nuclei of the epithelium. Using topical application of Acriflavine Hydrochloride followed by confocal microscopy, we could define morphological characteristics of each cellular layer of the normal human oral mucosa, building an atlas of histology-like images. Applying this technique to diseased oral tissue specimen, we were also able to accurately diagnose the presence of high-grade dysplasia through the increased cellularity and changes in nuclear morphological features. Objective measurement of cellular density by quantitative image analysis was a strong discriminant to differentiate between high-grade dysplasia and low-grade dysplasia lesions. CONCLUSIONS: Pending clinical investigation, real-time confocal microscopy may become a useful adjunct to detect precancerous lesions that are at high risk of cancer progression, direct biopsy and delineate excision margins.

Nuclear texture features for classifying benign vs. dysplastic or malignant squamous epithelium of the larynx.

OBJECTIVE: To search for nuclear features and feature combinations able to assess malignancy and premalignant changes on tissue sections of laryngeal squamous epithelium. STUDY DESIGN: A total of 139 lesions of benign changes (BC) (n = 44), epithelial dysplasias (ED) (n = 50) and invasive laryngeal cancer (LC) (n = 45) were retrieved from archival pathology specimens. The goal of this study was to identify the best features or feature combinations that discriminate BC from LC and also reflect the degree of ED. In order to verify the results on independent data, the groups were split into two separate subgroups, one for training and one for testing. RESULTS: On the test set of slides, the overall correct classification of BC vs. LC cases was 82% using only one feature, fractal2_area. This classification rate could be increased to 91% when a discriminant function based on 10 features was used. However, this gain was not significant. CONCLUSION: Fractal texture features can be used to assess malignancy on tissue sections as an alternative to DNA measurement. In this study feature combinations did not significantly improve classification rates.

Fiber-optic confocal microscopy using a spatial light modulator.

We present a novel fiber-optic confocal microscope in which the scanning operation is achieved by use of a spatial light modulator (SLM) to sequentially illuminate individual fibers or patterns of multiple fibers. Experimental images are presented, and the optical-sectioning capability of the device is demonstrated. The novel SLM-based system is more optically efficient, achieves higher contrast, and has improved optical-sectioning capabilities compared with those of other proposed instruments for confocal microendoscopy.

New method for detection of heart allograft rejection: validation of sensitivity and reliability in a rat heterotopic allograft model.

BACKGROUND: Patients with inflammatory heart muscle diseases would benefit from a safe, convenient, rapidly performed diagnostic technique with real-time results not involving tissue removal. We have performed a detailed evaluation of detection of heart allograft rejection by autofluorescence in a heterotopic abdominal rat heart allograft model ex vivo. METHODS AND RESULTS: Recipient rats with allograft (Lewis to Fisher 344; n=71) and isograft (Lewis to Lewis; n=33) hearts, treated with cyclosporine or untreated, were killed at days 2, 4, 7, 14, 21, 28, and 56 after transplant. Nontransplant controls with (n=24) or without (n=24) immunosuppressive therapy were also studied. When the rats were killed, autofluorescence spectra were acquired under blue-light excitation from midtransverse ventricular sections of native and transplanted hearts. Corresponding sections were then evaluated pathologically by a modified International Society for Heart and Lung Transplantation (ISHLT) grading schema. The spectral differences between rejecting and nonrejecting hearts were quantified by linear discriminant functions, producing scores that decreased progressively with increasing severity of tissue rejection. Mean+/-SD discriminant function scores were 2.9+/-1.6, 1.8+/-2.2, -0.1+/-2.8, -1.2+/-2.3, and -2.3+/-3.0 for isografts and allograft ISHLT grades 0, I, II, and III, respectively (Spearman rank-order correlation -0.6; P<0.001, test for trend). Cyclosporine had no detectable effect on the spectra. CONCLUSIONS: The correlation between changes in autofluorescence spectra and ISHLT rejection grade strongly supports the possibility of catheter-based, fluorescence-guided surveillance of rejection.

Image cytometry and chemoprevention in cervical cancer.

Of the approximately 60 million Pap smears performed in the United States in 1995, about 8% or 5 million will show cytology that is "not negative" (ASCUS, AGCUS, LSIL, HSIL, etc.). Possibly 15% or about 0.7 million of these cases will have positive follow-up by repeated Pap smears, colposcopy or biopsy. More than 4 million will be false-positive smears based on the reference standard of biopsy or repeated smears. If no treatment or medical intervention was offered to the 0.7 million cytologically and histologically positive cases, perhaps 20,000 (3%) would develop into invasive cancer. Of the original 5 million cytologically "not negative" cases, fewer than 0.5% have the potential to develop into invasive cancer. While considerable attention has been paid to false-negatives in Pap screening, the above considerations indicate that the cytological and histological criteria for assessing the malignant potential of "not negative" samples might benefit from some refinement. Until such refinement occurs, any chemoprevention studies in cervix face a formidable signal-to-noise problem--worse than 1:30. This paper presents data from quantitative image cytometry of cervical smears for assessing the malignant potential of various "not negative" cases. We have approached this in two ways--by analyzing dysplastic cell nuclei and by analyzing the nuclei of cytologically normal cells growing in the vicinity of the neoplastic lesion. In both cases, nuclear features describing the distribution of the DNA in the cell nuclei (especially texture features) are the discriminating factors. Future research into the objective assessment of malignant potential of "not negative" cases is outlined.

Detection of dysplasia and carcinoma in situ by ratio fluorometry.

Fluorescence bronchoscopy was performed in 82 volunteers recruited from occupational groups at risk of exposure to asbestos and/or diesel fumes to determine whether differences in tissue autofluorescence between normal and malignant bronchial tissues can be used to improve the sensitivity of standard fiberoptic bronchoscopy in detecting dysplasic and carcinoma in situ (CIS). This study consisted of 25 nonsmokers, 40 exsmokers, and 17 current smokers with mean ages of 52, 55, and 49 yr, respectively. Tissue autofluorescence was induced by a blue light from an He-Cd laser coupled to the illumination channel of the bronchoscope and analyzed by a ratiofluorometer. One or more sites of moderate or severe dysplasia were found in 12% of the exsmokers and current smokers but in none of the nonsmoker volunteers. CIS was found in two of the exsmokers. The sensitivity of fluorescence bronchoscopy (86%) was found to be 50% better than that of conventional white-light bronchoscopy (52%) in detecting dysplasia and CIS. Pre- and post-bronchoscopy sputum cytology failed to detect these precancerous lesions. Our results suggest that fluorescence bronchoscopy may be an important new method that can improve the ability to detect and localize precancerous and/or CIS lesions.