A Method for the Interpretation of Flow Cytometry Data Using Genetic Algorithms.

BACKGROUND: Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. SUBJECTS AND METHODS: Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. RESULTS: Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC) curve of 0.912. CONCLUSIONS: The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.

Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer.

We constructed a large-scale functional network model in Drosophila melanogaster built around two key transcription factors involved in the process of embryonic segmentation. Analysis of the model allowed the identification of a new role for the ubiquitin E3 ligase complex factor SPOP. In Drosophila, the gene encoding SPOP is a target of segmentation transcription factors. Drosophila SPOP mediates degradation of the Jun kinase phosphatase Puckered, thereby inducing tumor necrosis factor (TNF)/Eiger-dependent apoptosis. In humans, we found that SPOP plays a conserved role in TNF-mediated JNK signaling and was highly expressed in 99% of clear cell renal cell carcinomas (RCCs), the most prevalent form of kidney cancer. SPOP expression distinguished histological subtypes of RCC and facilitated identification of clear cell RCC as the primary tumor for metastatic lesions.

Classification of renal cell carcinoma based on expression of VEGF and VEGF receptors in both tumor cells and endothelial cells.

Recent development of antiangiogenic therapy for renal cell carcinoma (RCC) has significantly improved the treatment of these often refractory tumors. However, not all patients respond to therapy and assays for predicting outcome are needed. As a first step, we analyzed a retrospective cohort of tumors and assessed the ability of VEGF and VEGF receptors (VEGF-R1, -R2 and -R3) to classify tumors. We analyzed tissue microarrays containing 330 RCCs using a novel method of automated quantitative analysis of VEGF and VEGF-R expression by fluorescent immunohistochemistry. Expression of markers was separately quantified within three tissue components: tumor cells, endothelial cells and adjacent normal epithelium. VEGF and VEGF receptors were tightly coexpressed both within tumors and within adjacent normal cells (all P-values <0.001). Tumor cell expression of VEGF-R1 and -R2 was strongly and inversely correlated with vessel area (P<0.0001). Unsupervised hierarchical clustering classified tumors by coordinated expression of VEGF and VEGF-Rs. The distribution of clear cell and papillary tumors was not significantly different between clusters. Clusters with high expression of VEGF and VEGF-Rs in the tumor cells exhibited poor survival when compared with the other clusters on uni- and multivariable analysis. VEGF and VEGF receptors exhibit a complex pattern of coordinated expression in RCC. Clustering tumors by VEGF and VEGF-R in tissue components demonstrates distinct tumor phenotypes with different outcomes, and may provide a means for determining which tumors will respond to what antiangiogenic therapies.

Fine needle aspiration of poorly differentiated oxyphilic (Hürthle cell) thyroid carcinoma: a case report.

BACKGROUND: Poorly differentiated oxyphilic (Hürthle cell) carcinomas are a more recently described variant of poorly differentiated thyroid carcinoma and are characterized by a prominent Hürthle cell component in a solid or trabecular arrangement. Clinically, poorly differentiated oxyphilic carcinomas behave more aggressively as compared to classic Hürthle cell carcinomas, which have a predominantly follicular pattern. Although the histology of these rare thyroid tumors has been reported in the literature, the cytologic features on fine needle aspiration biopsy have not been described before. CASE: A 73-year-old man with a long history of radioactive iodine and levothyroxine therapy for multinodular goiter presented with a painful, rapidly expanding, 6-cm, left thyroid mass with aggressive radiologic features. Fine needle aspiration biopsy of the mass yielded extremely cellular smears with a dual population of medium-sized follicular cells and numerous Hürthle cells. Subsequent thyroidectomy confirmed the malignant nature of this Hürthle cell-rich tumor, warranting a diagnosis of poorly differentiated oxyphilic (Hürthle cell) thyroid carcinoma. CONCLUSION: Poorly differentiated oxyphilic thyroid carcinoma is an aggressive variant of Hürthle cell carcinomas and must enter the differential diagnosis when fine needle aspiration biopsy of a radiologically aggressive thyroid mass yields extremely hypercellular smears with a prominent Hürthle cell component.

Detection of malignancy in cytology specimens using spectral-spatial analysis.

Despite low sensitivity (around 60%), cytomorphologic examination of urine specimens represents the standard procedure in the diagnosis and follow-up of bladder cancer. Although color is information-rich, morphologic diagnoses are rendered almost exclusively on the basis of spatial information. We hypothesized that quantitative assessment of color (more precisely, of spectral properties) using liquid crystal-based spectral fractionation, combined with genetic algorithm-based spatial analysis, can improve the accuracy of traditional cytologic examination. Images of various cytological specimens were collected every 10 nm from 400 to 700 nm to create an image stack. The resulting data sets were analyzed using the Los Alamos-developed GENetic Imagery Exploitation (GENIE) package, a hybrid genetic algorithm that segments (classifies) images using automatically 'learned' spatio-spectral features. In an evolutionary fashion, GENIE generates a series of algorithms or 'chromosomes', keeping the one with best fitness with respect to a user-defined training set. First, we tested the system to determine if it could recognize malignant cells using artificial cytology specimens constructed to completely avoid the requirement for human interpretation. GENIE was able to differentiate malignant from benign cells and to estimate their relative proportions in controlled mixtures. We then tested the system on routine cytology specimens. When targeted to detect malignant urothelial cells in cytology specimens, GENIE showed a combined sensitivity and specificity of 85 and 95%, in samples drawn from two separate institutions over a span of 4 years. When trained on cases initially diagnosed as 'atypical' but with unequivocal follow-up by biopsy, surgical specimen or cytology, GENIE showed efficiency superior to the cytopathologist with respect to predicting the follow-up result in a cohort of 85 cases. We believe that, in future, this type of methodology could be used as an ancillary test in cytopathology, in a manner analogous to immunostaining, in those situations when a definitive diagnosis cannot be rendered based solely on the morphology.

Novel tyramide-based tyrosinase assay for the detection of melanoma cells in cytological preparations.

Fine-needle aspiration (FNA) has been used as a fast, minimally invasive, and reliable method for the evaluation of enlarged lymph nodes. However, there are some cases where the definitive diagnosis cannot be elicited with morphology alone, especially cases without a known primary lesion. Although immunocytochemical studies may be helpful in some situations, they are often complicated by nonspecific staining. Recently, a novel tyramide-based tyrosinase assay was developed. Since melanocytes, both benign and malignant, produce tyrosinase, we postulated that this assay could be useful as an in situ biochemical diagnostic test. We modified the Perkin Elmer TSA assay, a commercial assay based on tyramide, a tyrosine analog that is a substrate for tyrosinase, for use on air-dried cytological preparations. We validated the assay on cell lines, then tested a small series of melanoma and nonmelanoma cytology specimens. The YUGEN8 melanoma cell line was used to optimize the assay and it showed abundant reaction product, while HeLa cells served as a negative control. All melanoma cytology specimens were positive and all nonmelanoma specimens were negative. These results suggest that this simple, fast, and inexpensive assay is a sensitive and specific method for detection of melanoma cells in cytology specimens. This method may be a useful ancillary procedure for the resolution of challenging melanoma cases.

Diagnostic classification of urothelial cells in urine cytology specimens using exclusively spectral information.

BACKGROUND: Although cytologic evaluation of urine specimens is a standard procedure in the diagnosis and follow-up of bladder carcinoma, its sensitivity and specificity are low. Cytopathologic diagnoses are driven primarily by spatial relations or morphology. Although color enhances the pathologist's perception of the specimen, spectral information plays a minimal role in diagnostic processes. Recently, methods have been developed to capture and analyze spectral information from clinical specimens. In the current study, the authors determined the classification value of spectral information by testing its ability to discriminate between malignant and benign urothelial cells in cytology specimens. METHODS: Multiple images of benign urothelial cells (n = 39) and urothelial carcinoma cells (n = 35) were collected at serial wavelengths using a liquid crystal tunable optical filter and composited into a mosaic using ENVI (Environment for Visualizing Images) software. Through minimum noise fractionation and principal component analysis, the spectral information in the mosaic was compressed into a 29-dimensional scatter plot. The data generated were analyzed using visual and spectral end member extraction on both the original data set and a second independent data set (test set). RESULTS: One area of spectral clustering in the scatter plot segmented with carcinoma cells exclusively (100% specific), but was not present in every cell (approximately 50%), which may indicate that these spectral profiles are present in a subpopulation of malignant cells or at specific points of their cell cycle. Using ENVI algorithms, the authors found that a particular classification spectrum (end member 9) and its closest relatives identified malignant cell clusters, with a sensitivity and specificity that reached 82% and 81%, respectively. To validate this mechanism in a test set, a second mosaic comprised of 15 benign and 15 malignant clusters was analyzed using end member 9, resulting in a combined sensitivity and specificity of 73%. CONCLUSIONS: The results of the current study demonstrate that spectral information, in the complete absence of morphologic or spatial information, allows discrimination of benign and malignant urothelial cells in routine urine cytology specimens. The authors believe that this novel technology, combined with spatial analysis, has the potential to serve as an ancillary test for improved detection of bladder carcinoma.

Application of proteomic technologies to cytologic specimens. A review.

With the increasing use of molecular biology techniques in routine pathology practice, it seems reasonable that their application to cytologic specimens will become popular in the near future. Proteomics is a novel area of research that involves the global analysis of tissue proteins using diverse technologies, such as 2-D gel electrophoresis, mass spectrometry and bioinformatics. This review discusses recent applications of proteomic analysis to cytologic specimens as well as its potential in the diagnosis of neoplastic and nonneoplastic disease.

Effect of ethanol on glycerolipid and faty acid metabolism in Hep G2 human-hepatoma cells

It is well-known that ethanol alters fatty acid and glycerolipid metabolism in liver, but most of the studies have been deleloped on rats, so little known about the corresponding effects on human liver. We have chosen the Hep G2 human hepatoma cell line, which appears be an excellent in vitro model system. Cells were incubated in ethanol containing medium (0-400 mM) for 48 h. Incorporation and metabolism of radioactive substrates (14C(U) glycerol, [1-(14)C] palmitic acid and [1-(14)C] eicosatrienoic acid (n-6) were analyzed in cellular and conditioned medium lipids. Cellular growth rate and lipid composition of control and ethanol-treated cells were also studied. The results showed that ethanol inhibited logarithmic cellular growth rate in a concentration dependent manner, without affecting viability. Ethanol (400 mM) did not modify cellular major lipid composition except for an increase of cholesteryl esters, but produced a decrease in the proportions of myristic, palmitic and palmitoleic acids. Ethanol enchanced the incorporation of radioactive fatty acids into cellular glycerolipids but did not alter the rate of incorporation of 14C(U) glycerol. This was attributed to an isotopic solution of the radioactive glycerol as a result of increasde alfa-glycerophosphate biosynthesis. Incorporation of radioactive fatty acids and glycerol into conditioned medium glycerolipids were increased in cells incubated in presence of ethanol. The increased incorporation of 14C glycerol into conditioned medium together with a simultaneous diminuition in labeling cellular glycerides suggest that there would be a simulation of the export of these lipid classes to conditioned medium. Conversion of [1-(14)C] palmitic to oleic acid and eicosatrienoic to arachidonic acid were inhibited in 400 mM ethanol treated cells suggesting an inhibition of delta 9 and delta 5 desaturase activity.

Effect of ethanol on glycerolipid and faty acid metabolism in Hep G2 human-hepatoma cells

It is well-known that ethanol alters fatty acid and glycerolipid metabolism in liver, but most of the studies have been deleloped on rats, so little known about the corresponding effects on human liver. We have chosen the Hep G2 human hepatoma cell line, which appears be an excellent in vitro model system. Cells were incubated in ethanol containing medium (0-400 mM) for 48 h. Incorporation and metabolism of radioactive substrates (14C(U) glycerol, [1-(14)C] palmitic acid and [1-(14)C] eicosatrienoic acid (n-6) were analyzed in cellular and conditioned medium lipids. Cellular growth rate and lipid composition of control and ethanol-treated cells were also studied. The results showed that ethanol inhibited logarithmic cellular growth rate in a concentration dependent manner, without affecting viability. Ethanol (400 mM) did not modify cellular major lipid composition except for an increase of cholesteryl esters, but produced a decrease in the proportions of myristic, palmitic and palmitoleic acids. Ethanol enchanced the incorporation of radioactive fatty acids into cellular glycerolipids but did not alter the rate of incorporation of 14C(U) glycerol. This was attributed to an isotopic solution of the radioactive glycerol as a result of increasde alfa-glycerophosphate biosynthesis. Incorporation of radioactive fatty acids and glycerol into conditioned medium glycerolipids were increased in cells incubated in presence of ethanol. The increased incorporation of 14C glycerol into conditioned medium together with a simultaneous diminuition in labeling cellular glycerides suggest that there would be a simulation of the export of these lipid classes to conditioned medium. Conversion of [1-(14)C] palmitic to oleic acid and eicosatrienoic to arachidonic acid were inhibited in 400 mM ethanol treated cells suggesting an inhibition of delta 9 and delta 5 desaturase activity. (AU)