Quantification of epithelial volume by image processing applied to ovarian tumors

This paper describes an image analysis technique for automated estimation of the percentages of epithelium and stroma in (tumor) tissue. The program is evaluated on ovarian tumors of the serous, mucinous, and endometrioid type. From standard paraffin sections, stained with pararosanilin Feulgen and naphthol yellow, a blue-yellow image pair was recorded. The blue image was used for the determination of the total tissue area and the yellow image for the epithelial area. For the latter the image processing method is based on the fact that epithelial nuclei are generally more tightly packed than stromal nuclei. A structural approach was applied, in which the segmentation of the nuclei was based on the image contrast range in the density domain. The method has been tested with 78 image pairs from 19 ovarian tumors with varying degrees of malignancy. The area percentages, as assessed with image processing, were strongly correlated to control percentages, established by interactive morphometry (r = 0.98).     

Evaluation of the prognostic value of morphometric features and cellular DNA content in FIGO I ovarian cancer patients

Approximately 20% to 40% of the patients with a FIGO I ovarian tumor die within five years after the diagnosis. Morphologic studies (typing and grading) of the primary tumor are prognostically important, but poorly reproducible. Therefore, the prognostic value of more objective techniques, such as morphometry and flow cytometric (FCM) DNA determinations, were evaluated in 33 adequately staged FIGO I patients with at least a five-year follow-up. The overall five-year survival in the group was 64%. Three patient categories were defined on the basis of two easily measured morphometric features, the mitotic activity index (MAI) and the volume percentage epithelium (VPE), which an earlier study had proved to be significantly associated with prognosis. The five-year survival rates were 91% for 11 patients in category A (MAI less than 30 and VPE less than 65), 67% for 9 patients in category B (MAI less than 30 and VPE greater than or equal to 65) and 38% for 13 patients in category C (MAI greater than or equal to 30). FCM showed 25 of the tumors to be diploid and 8 to be aneuploid. The cellular DNA content was also of prognostic value: the five-year survival figures for patients with diploid and aneuploid tumors were 68% and 37%, respectively. Combination of the morphometric and FCM features showed that, in diploid tumors, the morphometric features have additional prognostic value: the diploid-tumor-patient survival rates in categories A, B and C were 91%, 63% and 50%, respectively. None of the eight patients with aneuploid tumors fell in the morphometrically favorable category A while seven were in category C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).     

Prognostic value of morphometry in acute lymphoblastic leukemia of childhood.

The prognostic value of morphometric features was studied in a group of 33 children with acute lymphoblastic leukemia (ALL) and compared with clinical and hematologic parameters. Air dried, May-Grünwald-Giemsa-stained specimens were prepared from iliac crest biopsies, and for each patient, 150 blasts and their nuclei were selected according to a stratified selection method and measured on a graphic tablet system. Univariate overall survival analysis showed the French-American-British (FAB) classification to be the strongest clinical parameter (P less than .0001). However, the significance was mainly due to the fact that both L3 cases died; the results for L1 and L2 were less satisfactory, with a survival rate (at 10 years) of 69% for the 26 L1 cases and 80% for the five L2 cases. The nuclear/cytoplasmic (N/C) ratio was the best morphometric feature (P less than .0001) and provided more satisfactory classification results than did FAB: only 2 of the 21 (10%) cases with N/C ratios greater than 0.90 died (7 and 9.5 years after the diagnosis, respectively), and 9 of the 12 (75%) cases with N/C ratios greater than or equal to 0.90 died. For recurrence-free survival analysis, essentially the same results were obtained. The N/C ratio retained its significant prognostic value after recurrence: 11 of the 15 patients with eventual recurrences died; 9 of them had an (original) N/C ratio less than or equal to 0.90. Three of the four recurring cases that survived after recurrence had an N/C ratio greater than 0.90 (P less than .03).(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.