Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissue.

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.     

DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine-Feulgen-stained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.     

Image cytometry of aneuploidy, growth fraction (MoAb Ki-67) and hormone receptors (ER, PR) immunocytochemical assays in breast carcinomas

DNA nuclear content was assessed in human breast carcinomas (n = 132) using image cytometry. Optical density histograms of Feulgen stained cell imprints from fresh tissue samples, subsequently frozen for immunocytochemical assays, were determined by the SAMBA system and used for the DNA index, the ploidy balance (PB) and the proliferation index (PI) computation. The three parameters were correlated to (i) histological data (tumour grade, vascular and/or lymph node invasion) and to (ii) growth fraction (Ki67), hormone receptor antigenic sites (ER, PR) and intramedullar (bone marrow) biopsies and anti-KL1-positive epithelial cells. It was shown that 57% of breast carcinomas were aneuploid. Aneuploidy PI significantly correlated to the criteria of poor prognosis such as high tumour grade, vascular and lymphatic invasion and to increased Ki67-positive cells, and the absence of or low ER and PR. Since image cytometry is easy to handle and perfectly suitable for current diagnostic practice in pathology departments, particularly for tumour cell ploidy assessment and standardized analysis of immunostaining procedures with morphological control of the preparation, we conclude that image cytometry, as performed with the SAMBA, must be regarded as a relevant tool for prognosis evaluation and therapy guidance in individual patients.     

A simple method to select specific tumor areas in paraffin blocks for cytometry using incident fluorescence microscopy

A simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopathological criteria is feasible using the adjacent section after hematoxylin-eosin staining as a diagnostic guideline. Once the position of the tumor area is determined, a small hollow bore, which is screwed in the objective holder, is pressed in the tissue. The depth is controlled by the microscrew of the microscope. After retraction of the bore, the block is removed, and a thick section is cut. The selected area can be processed for cytometry separately from the remaining tissue. The technique can be used either to enrich the sample to be analyzed with tumor cells or to analyze histopathologically different tumor compartments. Both flow and image cytometry can make use of this selection technique.     

Aptamers: versatile probes for flow cytometry

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.     

Cell cycle and apoptosis in tumours of oral cavity estimated by image cytometry

The aim of investigations was to examine whether diploid and aneuploid tumours of oral cavity differ in frequencies of occurrence of individual phases of cell cycle and of apoptosis. Investigations were carried out on 53 tumours using techniques of image cytometry. The studies were performed on properly isolated cells of tumours, which were stained using Feulgen method and analysed by image cytometry. Using a suitable computer programme, the percentages of cells present in individual phases of cell cycle and in apoptosis were estimated. No respective differences could be disclosed between diploid and aneuploid tumours. In addition, the ratios of the fraction of cells in S phase of the cell cycle and those in apoptosis were calculated and were found to carry certain prognostic significance in patients with squamous cell carcinomas of oral cavity.     

Intratumoural heterogeneity of nuclear DNA-content and proliferation in clear cell type carcinomas of the kidney.

Clear celled renal carcinomas (n = 37) were investigated by flow cytometry for intratumoural heterogeneity in DNA-ploidy and proliferation (S-phase rate). Using gross sections of the tumours, 178 regions of interest were selected and excised from the paraffin blocks. Of the tumours examined 30% (n = 11) were DNA-diploid and 70% (n = 26) were DNA-aneuploid. In six tumours (16%) homogenous DNA-aneuploidy was detected, and in 20 others (54%) there was intratumoural heterogeneity of DNA-content with a blend of either DNA-diploid and DNA-aneuploid regions (n = 16; 43%) or different aneuploid stemlines (n = 4; 11%). DNA-aneuploidy was present both in areas of the tumours composed of clear cells and in regions containing cells with cytoplasmatic eosinophilia. However, DNA-aneuploidy was correlated in a statistically highly significant manner with the degree of cytoplasmatic eosinophilia and the nuclear grading of tumour cells. The results were confirmed by comparative analysis of fresh-frozen and paraffin-embedded material. The DNA-aneuploid portions of the tumours, and the regions with increased cytoplasmatic eosinophilia, proved to have significantly higher S-phase rates than DNA-diploid and clear tumour cells. These results agreed well with the immunohistochemically determined percentage of Ki-67 (proliferation associated)-antigen positive cells. Our findings indicate that tumour cells with increased eosinophilia in renal cell carcinomas are distinct from real clear cells by virtue of their higher rates of aneuploidy and proliferative activity. These cells might therefore be regarded as a subclass with a more aggressive biological behaviour.     

Advances in the detection of ploidy differences in cancer by in situ hybridization.

Three main techniques allow the detection of changes in the cellular genomic content. The karyotyping procedure on metaphase spreads can give specific information on chromosome number and structural chromosome changes, but analyses are restricted to a limited number of chromosome spreads. Furthermore, cell culturing of (in particular solid) cancer specimens can result in selection of a minor tumour cell population with a high proliferative capacity. On the other hand, flow cytometry allows the analyses of large numbers of cells, but does not detect small variations in the DNA content or structural changes. The fluorescent in situ hybridization (FISH) procedure combines the advantages of the two former procedures, in that relatively large numbers of cells can be analysed easily and specific chromosomal changes can be detected.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

Screening and Identification of a Specific Binding Peptide to Ovarian Cancer Cells from a Phage-Displayed Peptide Library

To select specific binding peptides for imaging and detection of human ovarian cancer. The phage 12-mer peptide library was used to select specific phage clones to ovarian cancer cells. After four rounds of biopanning, the binding specificity of randomly selected phage clones to ovarian cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). DNA sequencing and homology analysis were performed on specifically bound phages. The binding ability of the selected peptides to SKOV3 cells was confirmed by fluorescence microscopy and flow cytometry. After four rounds of optimized biological panning, phage recovery was 34-fold higher than that of the first round, and the specific phage clones bound to SKOV3 cells were significantly enriched. A total of 32 positive phage clones were preliminarily identified by ELISA from 54 randomly selected clones, and the positive rate was 59.3%. S36 was identified as the clone with best affinity to SKOV3 cells via fluorescence microscopy and flow cytometry. A representative clone of OSP2, S36 is expected to be an effective probe for diagnosis and treatment of ovarian cancer.

     

Topography of genetic loci in tissue samples: towards new diagnostic tool using interphase FISH and high-resolution image analysis techniques.

Using single and dual colour fluorescence in situ hybridisation (FISH) combined with image analysis techniques the topographic characteristics of genes and centromeres in nuclei of human colon tissue cells were investigated. The distributions of distances from the centre-of-nucleus to genes (centromeres) and from genes to genes (centromeres to centromeres) were studied in normal colon tissue cells found in the neighbourhood of tumour samples, in tumour cell line HT-29 and in promyelocytic HL-60 cell line for comparison. Our results show that the topography of genetic loci determined in 3D-fixed cell tissue corresponds to that obtained for 2D-fixed cells separated from the tissue. The distributions of the centre-of-nucleus to gene (centromere) distances and gene to gene (centromere to centromere) distances and their average values are different for various genetic loci but similar for normal colon tissue cells, HT-29 colon tumour cell line and HL-60 promyelocytic cell line. It suggests that the arrangement of genetic loci in cell nucleus is conserved in different types of human cells. The investigations of trisomic loci in HT-29 cells revealed that the location of the third genetic element is not different from the location of two homologues in diploid cells. We have shown that the topographic parameters used in our experiments for different genetic elements are not tissue or tumour specific. In order to validate high-resolution cytometry for oncology, further investigations should include more precise parameters reflecting the state of chromatin in the neighbourhood of critical oncogenes or tumour suppresser genes.     

Nuclear image analysis of immunohistochemically stained cells in breast carcinomas

Hitherto, the relationship between malignancy-associated morphological features in single tumour cells and the expression of markers indicating functional properties of these cells remained widely unknown. This study was aimed at describing differences in the size, shape and chromatin structure between tumour cells with different marker expression for progesterone receptors (PgR) and p53. Two series of breast cancers, consisting of 50 PgR-positive, and 39 p53-negative and 49 p53-positive mammary carcinomas, were investigated. The immunohistochemical staining was performed on paraffin sections using 3-amino-9-ethylcarbazole as the chromogenic substrate. By means of a cytometry workstation equipped with a computer-controlled motorised scanning stage, about 500 positive and negative tumour cells in each case were localised in the microscope and categorised by a scoring system for their staining intensity. After destaining, the tissue sections were Feulgen-stained. Then, all the tumour cells were relocated automatically and analysed by high resolution image cytometry. Among the numerous size, shape, and texture features used in the system, several variables of the nuclear contour and chromatin structure were found to be significantly different between the positive and negative tumour cell populations. Nuclei without PgR had more malignancy-associated morphological features than PgR-positive cells. Whereas p53-negative nuclei had a higher degree of regularity, their positive counterparts exhibited higher DNA ploidy values.     

An image analysis method for assessment of prognostic risk in prostate cancer: a pilot study

Fully automated computerized image analysis at medium resolution (1 micron per pixel space) was applied in a study of 17 patients with stage D1 prostate cancer. For this pilot study, patients were selected on the basis of very good or very poor outcome. This selection was made in the hope of identifying morphometric features that are useful in prognostic assessment. Nine patients with good outcome were alive after 7 or more years of follow-up and eight patients with poor prognosis were dead of disease in less than 3 years. All patients were treated with 125I seed implantation to the prostate and pelvic lymph node dissection. Hormone therapy was not administered until the time of distant failure. Routine hematoxylin and eosin tissue sections of lymph nodal tissue bearing metastatic neoplasm were used for this analysis. A minimum of eight scenes per case was analysed. Of 50 measured parameters on each cluster, five (gray level distribution, number of cell clusters per scene, bending energy, average cluster area and cluster polarity) were useful to distinguish patients with good outcome from those with a poor outcome. Thirteen of the 17 patients were correctly classified by image analysis (P = 0.044, Fischer's exact test). By comparison, flow cytometry of the identical tissue samples correctly classified 14 of 17 patients (diploid, good outcome; aneuploid, poor outcome; P = 0.009). Only one patient was incorrectly classified by both image analysis and flow cytometry, implying a complementary prognostic role for the two methods. The encouraging result, successful identification of useful morphometric features, justifies a larger study of unselected patients.     

A cell-based single-stranded DNA aptamer specifically targets gastric cancer

Gastric cancer is one of the most prevailing cancers with high morbidity and mortality. Limitations in the current diagnosis and therapy, specially lacking of specific molecular therapeutic targets, ask for the development of new strategies. Aptamer, a newly developed adaptive molecule, could be used in clinical detection and therapy because of its high affinity and specificity. As no aptamer has ever been developed in preventing gastric cancer so far, we were the first who cloned such an aptamer specifically targeting gastric cancer. The aptamer was selected by systematic evolution of ligands by exponential enrichment with gastric cancer cell-line HGC-27 as target cell line and immortalized gastric epithelial cell-line GES-1 as control cell line. The affinity and specificity of candidate aptamers were examined by flow cytometry, confocal imagining and aptamer-based histochemistry staining. After 19 cycles of systematic evolution of ligands by exponential enrichment and subsequent cloning and sequencing, an aptamer with the highest affinity and specificity (nominated as AGC03) among candidates was screened out from a random single-stranded DNA pool. Moreover, AGC03 could not only specifically bind to gastric cancer cells (the equilibrium dissociation constant value was 16.49 ± 0.40 nM) in vitro, but also recognize cancer cells in human cancer tissue. Our most important finding is that AGC03 could even be internalized into cells automatically. In conclusion, we obtained a novel aptamer specifically targeting gastric cancer, which is an effective tool for both gastric cancer diagnosis and drug delivery.     

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY

A new method for the evaluation of cell production rates combining flow cytometry (FCM) and the stathmokinetic method using vineristine sulphate (VS) has been used for the analysis of three aneuploid ascites tumours at different stages of growth. Using this technique it was possible to estimate the well-known decrease in cell production rates of ageing ascites tumours. The percentage of normal host cells in the aneuploid tumours studied was easily determined by FCM prior to the calculation of the tumour cell-production rates. A correlation was found between the percentage of tumour cells in the S phase and the tumour cell-production rate. This correlation is probably explained by the gradual transfer of proliferating cells in S phase to resting G₁ and G₂ phases with increasing tumour age.     

Image and flow cytometric analysis of DNA content in breast cancer. Relation to estrogen receptor content and lymph node involvement

A comparative flow-cytometric and image-cytometric study was performed on 166 human breast cancers. Parallel measurements of 67 cases showed a good correlation between the DNA indices measured with each of the techniques. However, minor ploidy abnormalities were detected with flow cytometry. Only with this technique about 70% of the tumors appeared to be aneuploid. DNA profiles obtained with image cytometry frequently contained DNA values above the modal G2 value of the tumor. At least part of these broadly scattered DNA profiles resulted from multiploid tumors, as was found with flow cytometry. The mean percentage of cells with DNA contents exceeding the modal G0G1 value in image-cytometric DNA profiles (non- G0G1 cells) appeared to be highest in aneuploid lymph-node-positive (N+) tumors and lowest in near-diploid lymph-node-negative (N-) tumors. Near-diploid tumors were more frequently (70%) estrogen receptor positive (ER+) than were aneuploid tumors (50%), whereas highly aneuploid tumors with low or negative ER contents tended to be N+. Tumors with a high percentage of non- G0G1 cells value were predominantly estrogen-receptor negative (ER-).     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

Immunohistologic techniques for detecting the glycolipid Gb3 in the mouse kidney and nervous system

Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb(3) was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.