Triple-clad W-type fiber mitigates multipath artifacts in multimodal optical coherence tomography.

Multimodal endoscopic optical coherence tomography (OCT) can be implemented with double-clad fiber by using the presumed single-mode core for OCT and the higher numerical aperture cladding for a secondary modality. However, the quality of OCT in double-clad fiber (DCF) based systems is compromised by the introduction of multipath artifacts that are nt present in single-mode fiber OCT systems. Herein, the mechanisms for multipath artifacts in DCF are linked to its modal contents using a commercial software package and experimental measurement. A triple-clad W-type fiber is proposed as a method for achieving multimodal imaging with single-mode quality OCT in an endoscopic system. Simulations of the modal contents of a W-type fiber are compared to DCF and single-mode fiber. Finally, a W-Type fiber rotary catheter is used in a DCF-based endoscopic OCT and autofluorescence imaging (AFI) system to demonstrate multipath artifact free OCT and AFI of a human fingertip.

Multipath artifacts enable angular contrast in multimodal endoscopic optical coherence tomography.

Multipath artifacts are inherent to double-clad fiber based optical coherence tomography (OCT), appearing as ghost images blurred in the A-line direction. They result from the excitation of higher-order inner-cladding modes in the OCT sample arm which cross-couple into the fundamental mode at discontinuities and thus are detected in single-mode fiber-based interferometers. Historically, multipath artifacts have been regarded as a drawback in single fiber endoscopic multimodal OCT systems as they degrade OCT quality. In this work, we reveal that multipath artifacts can be projected into high-quality two-dimensional en face images which encode high angle backscattering features. Using a combination of experiment and simulation, we characterize the coupling of Mie-range scatterers into the fundamental image (LP01 mode) and higher-order image (multipath artifact). This is validated experimentally through imaging of microspheres with an endoscopic multimodal OCT system. The angular dependence of the fundamental image and higher order image generated by the multipath artifact lays the basis for multipath contrast, a ratiometric measurement of differential coupling which provides information regarding the angular diversity of a sample. Multipath contrast images can be generated from OCT data where multipath artifacts are present, meaning that a wealth of clinical data can be retrospectively examined.

Economic evaluation of DNA ploidy analysis vs liquid-based cytology for cervical screening.

BACKGROUND: DNA ploidy analysis involves automated quantification of chromosomal aneuploidy, a potential marker of progression toward cervical carcinoma. We evaluated the cost-effectiveness of this method for cervical screening, comparing five ploidy strategies (using different numbers of aneuploid cells as cut points) with liquid-based Papanicolaou smear and no screening. METHODS: A state-transition Markov model simulated the natural history of HPV infection and possible progression into cervical neoplasia in a cohort of 12-year-old females. The analysis evaluated cost in 2012 US$ and effectiveness in quality-adjusted life-years (QALYs) from a health-system perspective throughout a lifetime horizon in the US setting. We calculated incremental cost-effectiveness ratios (ICERs) to determine the best strategy. The robustness of optimal choices was examined in deterministic and probabilistic sensitivity analyses. RESULTS: In the base-case analysis, the ploidy 4 cell strategy was cost-effective, yielding an increase of 0.032 QALY and an ICER of $18 264/QALY compared to no screening. For most scenarios in the deterministic sensitivity analysis, the ploidy 4 cell strategy was the only cost-effective strategy. Cost-effectiveness acceptability curves showed that this strategy was more likely to be cost-effective than the Papanicolaou smear. CONCLUSION: Compared to the liquid-based Papanicolaou smear, screening with a DNA ploidy strategy appeared less costly and comparably effective.

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.

Autofluorescence imaging can identify preinvasive or clinically occult lesions in fallopian tube epithelium: a promising step towards screening and early detection.

BACKGROUND: Optical imaging systems are robust, portable, relatively inexpensive, and have proven utility in detecting precancerous lesions in the lung, esophagus, colon, oral cavity and cervix. We describe the use of light-induced endogenous fluorescence (autofluorescence) in identifying preinvasive and occult carcinomas in ex vivo samples of human fallopian tube (FT) epithelium. METHODS: Women undergoing surgery for an i) ovarian mass, ii) a history suggestive of hereditary breast-ovarian cancer, or iii) known serous ovarian cancer following neoadjuvant chemotherapy (NAC) were approached for informed consent. Immediately following surgery, FT's were photographed in reflectance and fluorescence at high resolution. Images included: (1) white-light reflectance of luminal/epithelial surface; (2) narrow-band green reflectance (570 nm) (3) green autofluorescence (405/436 nm excitation); and (4) blue autofluorescence (405 nm excitation). Areas revealing a loss of natural tissue fluorescence or marked increase in tissue microvasculature were recorded and compared to final histopathologic diagnosis (SEE-FIM protocol). RESULTS: Fifty-six cases involving one or both fallopian tubes underwent reflectance and fluorescence visualization. Nine cases were excluded, either secondary to non-ovarian primary pathology (7) or excessive trauma (2) rendering tissue interpretation impossible. Of the 47 cases remaining, there were 11 high grade serous (HGS) and 9 non-serous ovarian carcinomas undergoing primary debulking surgery, 5 serous carcinomas having received NAC, 8 benign ovarian tumors, and 14 women undergoing risk-reducing bilateral salpingo-oophorectomy (RRBSO). Methodology was feasible, efficient, and reproducible. TIC or carcinoma was identified in 7/11 HGS, 3/5 NAC, and 1/14 RRBSO. Optical images were reviewed to determine test positive or negative based on standardized criteria. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for the entire cohort (73%; 83%; 57%; 91%) and in a subgroup that excluded non-serous histology (87.5%; 92%; 78%; 96%). CONCLUSIONS: Abnormal FT lesions can be identified using ex vivo optical imaging technologies. With this platform, we will move towards genomic interrogation of identified lesions, and developing in vivo screening modalities via falloposcopy.

DNA amplification is a ubiquitous mechanism of oncogene activation in lung and other cancers.

Chromosomal translocation is the best-characterized genetic mechanism for oncogene activation. However, there are documented examples of activation by alternate mechanisms, for example gene dosage increase, though its prevalence is unclear. Here, we answered the fundamental question of the contribution of DNA amplification as a molecular mechanism driving oncogenesis. Comparing 104 cancer lines representing diverse tissue origins identified genes residing in amplification 'hotspots' and discovered an unexpected frequency of genes activated by this mechanism. The 3431 amplicons identified represent approximately 10 per hematological and approximately 36 per epithelial cancer genome. Many recurrently amplified oncogenes were previously known to be activated only by disease-specific translocations. The 135 hotspots identified contain 538 unique genes and are enriched for proliferation, apoptosis and linage-dependency genes, reflecting functions advantageous to tumor growth. Integrating gene dosage with expression data validated the downstream impact of the novel amplification events in both cell lines and clinical samples. For example, multiple downstream components of the EGFR-family-signaling pathway, including CDK5, AKT1 and SHC1, are overexpressed as a direct result of gene amplification in lung cancer. Our findings suggest that amplification is far more common a mechanism of oncogene activation than previously believed and that specific regions of the genome are hotspots of amplification.

Cytology microarrays.

The use of high throughput genetic and expression platforms are generating many candidate diagnostic markers and therapeutic targets for a wide variety of clinical conditions. Tissue microarrays can be used for the evaluation of the utility of many of these markers. However, tissue microarrays can suffer from the limitations associated with sampling and sectioning tissues. We introduce a novel microarray technique based on cell suspensions. Multiple slides can be made, all of which are equally representative of the initial sample. A robotic device was designed that can deposit 60 distinct spots of cytological material on a glass slide. Each spot of cells deposited in this manner may correspond to a unique source. Controlling the number of cells per spot, their distribution within the spot and the size of the spot can be achieved by modifying the viscosity of the cell solution or regulating the amount of fluid deposited. A fully automated analysis of quantitatively stained microarray samples has been performed to quantify the number of cells per spot, the size of spots and the DNA amount per cell in each spot. The reproducibility of these parameters was found to be high.

Differential disruption of cell cycle pathways in small cell and non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related mortality in the world, with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) comprising the two major cell types. Although these cell types can be distinguished readily at the histological level, knowledge of their underlying molecular differences is very limited. In this study, we compared 14 SCLC cell lines against 27 NSCLC cell lines using an integrated array comparative genomic hybridisation and gene expression profiling approach to identify subtype-specific disruptions. Using stringent criteria, we have identified 159 of the genes that are responsible for the different biology of these cell types. Sorting of these genes by their biological functions revealed the differential disruption of key components involved in cell cycle pathways. Our novel comparative combined genome and transcriptome analysis not only identified differentially altered genes, but also revealed that certain shared pathways are preferentially disrupted at different steps in these cell types. Small cell lung cancer exhibited increased expression of MRP5, activation of Wnt pathway inhibitors, and upregulation of p38 MAPK activating genes, while NSCLC showed downregulation of CDKN2A, and upregulation of MAPK9 and EGFR. This information suggests that cell cycle upregulation in SCLC and NSCLC occurs through drastically different mechanisms, highlighting the need for differential molecular target selection in the treatment of these cancers.

Genomic and gene expression profiling of minute alterations of chromosome arm 1p in small-cell lung carcinoma cells.

Genetic alterations occurring on human chromosome arm 1p are common in many types of cancer including lung, breast, neuroblastoma, pheochromocytoma, and colorectal. The identification of tumour suppressors and oncogenes on this arm has been limited by the low resolution of current technologies for fine mapping. In order to identify genetic alterations on 1p in small-cell lung carcinoma, we developed a new resource for fine mapping segmental DNA copy number alterations. We have constructed an array of 642 ordered and fingerprint-verified bacterial artificial chromosome clones spanning the 120 megabase (Mb) 1p arm from 1p11.2 to p36.33. The 1p arm of 15 small-cell lung cancer cell lines was analysed at sub-Mb resolution using this arm-specific array. Among the genetic alterations identified, two regions of recurrent amplification emerged. They were detected in at least 45% of the samples: a 580 kb region at 1p34.2-p34.3 and a 270 kb region at 1p11.2. We further defined the potential importance of these genomic amplifications by analysing the RNA expression of the genes in these regions with Affymetrix oligonucleotide arrays and semiquantitative reverse transcriptase-polymerase chain reaction. Our data revealed overexpression of the genes HEYL, HPCAL4, BMP8, IPT, and RLF, coinciding with genomic amplification.

A Java application for tissue section image analysis.

The medical industry has taken advantage of Java and Java technologies over the past few years, in large part due to the language's platform-independence and object-oriented structure. As such, Java provides powerful and effective tools for developing tissue section analysis software. The background and execution of this development are discussed in this publication. Object-oriented structure allows for the creation of "Slide", "Unit", and "Cell" objects to simulate the corresponding real-world objects. Different functions may then be created to perform various tasks on these objects, thus facilitating the development of the software package as a whole. At the current time, substantial parts of the initially planned functionality have been implemented. Getafics 1.0 is fully operational and currently supports a variety of research projects; however, there are certain features of the software that currently introduce unnecessary complexity and inefficiency. In the future, we hope to include features that obviate these problems.

A pilot study for a screening trial of cervical fluorescence spectroscopy.

Fluorescence spectroscopy is a promising technology for detection of epithelial precancers and cancers. In preparation for a multicenter phase II screening trial, a pilot trial was conducted to test data collection and patient examination procedures, use data forms, time procedures, and identify problems with preliminary data analysis. Women 18 years of age and older underwent a questionnaire, a complete history, and a physical examination, including a pan-colposcopy of the lower genital tract. A fiber-optic probe measured fluorescence excitation-emission matrices at 1-3 cervical sites for 58 women. The data collection procedures, data forms, and procedure times worked well, although collection times for all the clinical data take an average of 28 min. The clinical team followed procedures well, and the data could be retrieved from the database at all sites. The multivariate analysis algorithm correctly identified squamous normal tissue 99% of the time and columnar normal tissue only 7%. The assessment of ploidy from monolayer samples was not accurate in this small sample. The study was successful as a pilot trial. We learned who participated, who withdrew, how often abnormalities were present, and that algorithms that have worked extremely well in previous studies do not work as well when a few study parameters are changed. The current algorithm for diagnosis identified squamous normal tissue very accurately and did less well for columnar normal tissue. Inflammation may be an explanation for this phenomenon. Fluorescence spectroscopy is a promising technology for the detection of epithelial precancers and cancers. The screening trial of fluorescence and reflectance spectroscopy was successful.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

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.

Therapeutic value of eptifibatide at community hospitals transferring patients to tertiary referral centers early after admission for acute coronary syndromes. PURSUIT Investigators.

OBJECTIVES: We aimed to evaluate the benefits of the glycoprotein (GP) IIb/IIIa antagonist, eptifibatide, after patients with acute coronary syndromes (ACS) were admitted to hospitals that approach revascularization for ACS through early transfer to tertiary referral centers. BACKGROUND: Across a variety of hospital settings, GP IIb/IIIa inhibition, after patients were admitted to the hospital for non-ST segment elevation ACS, is associated with a reduction in death or myocardial infarction (MI) before and during a percutaneous coronary intervention. METHODS: The outcomes of 429 patients from 153 sites in the Platelet glycoprotein IIb/IIIa in unstable angina: Receptor Suppression Using Integrilin Therapy (PURSUIT) trial, who were transferred during study drug infusion ("transfer patients"), were compared with those of 1,987 patients who either remained in the hospital at those sites or were transferred after study drug termination ("nontransfer patients"). RESULTS: The baseline characteristics of transfer and nontransfer patients were similar. Patients receiving eptifibatide were transferred less frequently than those receiving placebo (16% vs. 20%, p = 0.014). Transfer patients underwent more procedures and experienced a greater 30-day incidence of death or MI, as compared with nontransfer patients (21% vs. 12%, p = 0.001). Eptifibatide was associated with a reduction in death or MI through 30 days, independent of transfer status (2.5% absolute reduction), as well as for those transferred (5.5% absolute reduction). CONCLUSIONS: For patients with ACS admitted to community hospitals, eptifibatide is associated with a reduced need for transfer and improved clinical outcomes.

Specific changes of chromatin structure in nuclei of normal epithelium adjacent to laryngeal squamous cell carcinoma. A preliminary study of 82 cases.

The aim of this study was to confirm the existence of specific nuclear texture feature alterations of histologically normal epithelial borders nearby invasive laryngeal cancer (NC). Paraffin sections of NC and of chronic inflammations unrelated to cancer (CI) were analysed for nuclear texture and for integrated optical density (IOD-index) and were compared to normal epithelium of patients without evidence of cancer (NE). Several discriminant functions based on nuclear texture features were trained to separate different subgroups. As the most important result, specific nuclear texture feature shifts were only found in NC with high-density lymphocytic stroma infiltrate (NC+). Classification of nuclei of NE versus NC+ was correct in 70%. The same classifier was correct in only 58% when nuclei of NE were classified versus CI. We also found lower values of IOD-Index within the NC+ group when compared to NE (p < 0.001).

Detection and localization of early lung cancer by fluorescence bronchoscopy.

BACKGROUND: Curative therapy is available for patients with Stage 0 lung carcinoma, with a >90% 5-year survival rate. Promising chemopreventive agents also are under investigation currently to reduce the risk of lung carcinoma in high risk populations. However, preinvasive bronchial lesions (moderate to severe dysplasia and carcinoma in situ) are very small and thin. They are difficult to localize by conventional white-light bronchoscopy. Fluorescence bronchoscopy is a new diagnostic tool for the detection of these preinvasive lesions. METHODS: The data on the use of fluorescence bronchoscopy to detect and localize preinvasive lesions in current heavy smokers and in former smokers at the British Columbia Cancer Agency as well as the worldwide experience cited in MEDLINE, Index Medicus, and Deutsches Institut fur Medizinische Dokumentation und Information (Cologne, Germany) comparing white-light and fluorescence bronchoscopy using the lung imaging fluorescence endoscope (LIFE)-Lung device (Xillix Technologies Corp., Richmond, British Columbia, Canada) were reviewed. RESULTS: Among current heavy smokers and former smokers with sputum atypia, the prevalence of carcinoma in situ was 1.6%. Moderate or severe dysplasia was found in another 19%. The preinvasive lesions were found to be small: 55% measured < or = 1.5 mm in greatest dimension. Over 1000 cases have been reported in the literature between 1994 and 1999. Overall, 40% of the preinvasive lesions were detected by white-light bronchoscopy alone. The addition of fluorescence bronchoscopy increased the detection rate to an average of 80%. CONCLUSIONS: Preinvasive lesions, especially dysplastic lesions, are small. They are difficult to detect and localize by white-light bronchoscopy. Fluorescence bronchoscopy improves the detection rate. It is an important part of the armamentarium in the overall management of early lung cancer.

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.

The complete DNA sequence of myxoma virus.

Myxomatosis in European rabbits is a severely debilitating disease characterized by profound systemic cellular immunosuppression and a high rate of mortality. The causative agent, myxoma virus, is a member of the poxvirus family and prototype of the Leporipoxvirus genus. As a major step toward defining the genetic strategies by which the virus circumvents host antiviral responses, the genomic DNA sequence of myxoma virus, strain Lausanne, was determined. A total of 171 open reading frames were assigned to cover the 161.8-kb genome, including two copies each of the 12 genes that map within the 11.5-kb terminal inverted repeats. Database searches revealed a central core of approximately 120 kb that encodes more than 100 genes that exhibit close relationships to the conserved genes of members of other poxvirus genera. Open reading frames with predicted signal sequences, localization motifs, or homology to known proteins with immunomodulatory or host-range functions were examined more extensively for predicted features such as hydrophobic regions, nucleic acid binding domains, ankyrin repeats, serpin signatures, lectin domains. and structural cysteine spacings. As a result, several novel, potentially immunomodulatory proteins have been identified, including a family with multiple ankyrin-repeat domains, an OX-2 like member of the neural cell adhesion molecule family, a third myxoma serpin, a putative chemokine receptor fragment, two natural killer receptor-like species, and a variety of species with domains closely related to diverse host immune regulatory proteins. Coupled with the genomic sequencing of the related leporipoxvirus Shope fibroma virus, this work affirms the existence of a conserved complement of poxvirus-specific core genes and expands the growing repertoire of virus genes that confer the unique capacity of each poxvirus family member to counter the immune responses of the infected host.