Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients.

BACKGROUND: Current approaches for detecting circulating tumour cells (CTCs) in blood are dependent on CTC enrichment and are based either on surface epithelial markers on CTCs or on cell size differences. The objectives of this study were to develop and characterise an ultrasensitive multiplex fluorescent RNA in situ hybridisation (ISH)-based CTC detection system called CTCscope. This method detects a multitude of tumour-specific markers at single-cell level in blood. METHODS: Healthy blood samples spiked with tumour cell lines were used as a model system for the development and initial characterisation of CTCscope. To demonstrate the feasibility of CTC detection in patient blood, duplicate blood samples were drawn from 45 metastatic breast cancer patients for analysis by CTCscope and the CellSearch system. The association of CTCs with the tumour marker CA15-3 and progression-free survival (PFS) were assessed. RESULTS: CTCscope detected CTC transcripts of eight epithelial markers and three epithelial-mesenchymal-transition (EMT) markers for increased sensitivity. CTCscope was used to detect CTCs with minimal enrichment, and did not detect apoptotic or dead cells. In patient blood samples, CTCs detected by CellSearch, but not CTCscope, were positively correlated with CA15-3 levels. Circulating tumour cells detected by either CTCscope or CellSearch predicted PFS (CTCscope, HR (hazard ratio) 2.26, 95% CI 1.18-4.35, P=0.014; CellSearch, HR 2.50, 95% CI 1.27-4.90, P=0.008). CONCLUSION: CTCscope offers unique advantages over existing CTC detection approaches. By enumerating and characterising only viable CTCs, CTCscope provides additional prognostic and predictive information in therapy monitoring.

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy.

BACKGROUND: The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors. METHODS: Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA). RESULTS: The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047). CONCLUSION: Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer.

Molecular epidemiology of Campylobacter jejuni in broiler flocks using randomly amplified polymorphic DNA-PCR and 23S rRNA-PCR and role of litter in its transmission.

Poultry has long been cited as a reservoir for Campylobacter spp., and litter has been implicated as a vehicle in their transmission. Chicks were raised on litter removed from a broiler house positive for Campylobacter jejuni. Litter was removed from the house on days 0, 3, and 9 after birds were removed for slaughter. Chicks were raised on these three litters under controlled conditions in flocks of 25. None of these birds yielded C. jejuni in their cecal droppings through 7 weeks. Two successive flocks from the same Campylobacter-positive broiler house were monitored for Campylobacter colonization. Campylobacter jejuni prevalence rates were determined for each flock. Randomly amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates. A high prevalence (60%) of C. jejuni in flock 1 coincided with the presence of an RAPD profile not appearing in flock 2, which had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was used to determine if strains with different RAPD profiles and different prevalence rates contained different 23S sequences. RAPD profiles detected with higher prevalence rates contained a spacer in the 23S rRNA region 100% of the time, while RAPD profiles found with lower prevalence rates contained an intervening sequence less than 2% of the time. Data suggest varying colonizing potentials of different RAPD profiles and a source other than previously used litter as a means of transmission of C. jejuni. These molecular typing methods demonstrate their usefulness, when used together, in this epidemiologic investigation.

Minoxidil: inhibition of proliferation of keratinocytes in vitro.

Previous studies have suggested that minoxidil stimulates growth of keratinocytes, possibly in a manner similar to the action of epidermal growth factor. Using both a short-term assay, thymidine incorporation, and a longer term assay, cell counting, to assess proliferative growth, we tested the activity of minoxidil in human keratinocyte cultures grown in 0.1 mM Ca(++). Minoxidil failed to stimulate growth in these assays. At concentrations of 5-10 micrograms per ml, minoxidil showed half-maximal inhibition of both EGF- and placental extract-stimulated thymidine incorporation. Minoxidil also inhibited proliferative growth in the presence or absence of placental extract. Direct measurement of the ability of minoxidil to compete for binding to the EGF receptor indicated that minoxidil probably does not bind to the EGF receptor. Minoxidil was not toxic, as keratinocytes continued to survive and grow, although at a slower rate, in the presence of minoxidil.

Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis.

Trichohyalin, a protein contained in granules in the cells of the hair-follicle inner root sheath and in the medulla of the hair shaft, has been purified previously from sheep hair bulbs and is also a major protein of filiform papillae of tongue epithelium. Polyclonal affinity-purified antibodies and a monoclonal antibody raised to purified pig tongue trichohyalin both stained the inner root sheath of hair follicles and the medulla of hair fibers and identified human trichohyalin as a single 220-kDa band on immunoblots of human hair bulb proteins. These antibodies were used to examine human epidermis by immunofluorescence and immunoblotting. The antibodies decorate granules in cells in the granular layer and stratum corneum of non-hair-bearing human skin, and immunoblots identify a protein in epidermis comigrating with trichohyalin from human hair and human tongue epithelium. Absorption of antibody to trichohyalin on a trichohyalin affinity column abrogated staining of the epidermis and the bands on the immunoblots. Trypsin-separated epidermis contained 220 and 160 kDa bands identified as trichohyalin, but epidermis shaved from skin and quickly frozen showed only a single 220-kDa band, indicating that the 160-kDa protein was generated by proteolysis. Double immunofluorescence for trichohyalin and filaggrin showed that some cells containing filaggrin also contain trichohyalin. These studies show that trichohyalin is not limited to hair and tongue but is present in isolated cells in the granular layer and stratum corneum of normal epidermis.

Stimulation of growth of keratinocytes by basic fibroblast growth factor.

Numerous heparin-binding growth factors active in different types of cells have recently been shown to belong to the family of fibroblast growth factors. Because these factors are active in some types of epithelial cells, we tested the activity of basic fibroblast growth factor (bFGF) from bovine brain in human keratinocyte cultures. bFGF stimulated thymidine incorporation and cellular proliferation in these cultures with half-maximal activity at approximately 60 pg/ml (4 X 10(-12) M). Stimulation of thymidine incorporation was associated with increased nuclear labeling after 22 h in the presence of bFGF under the same conditions used in the thymidine incorporation assay. bFGF was nearly as effective as epidermal growth factor (EGF) in stimulating keratinocyte growth and substantially less effective than crude placental extract, and was not additive with EGF in stimulating thymidine incorporation or proliferation of cells. The findings indicate that bFGF is a potent growth factor for keratinocytes.

Keratinocyte growth-promoting activity from human placenta.

Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25-500 micrograms/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth-promoting activity in bovine hypothalamic extract, the activity is non-dialyzable and is destroyed at 100 degrees C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth-promoting properties for epithelium.

Spreading and enhanced motility of human keratinocytes on fibronectin.

Soluble human plasma fibronectin or collagen types I or IV, when preincubated with tissue culture plastic dishes, were effective spreading agents for cultured human keratinocytes and increased spreading in a time-and concentration-dependent manner. Spreading on fibronectin, but not on type IV collagen, was inhibited by antifibronectin; therefore, the contribution of fibronectin to the spreading activity of the natural matrix produced by keratinocytes could not be determined using antifibronectin. Fibronectin mediated spreading at both high (1.1 mM) and low (0.1 mM) Ca++ concentrations, and spreading was not altered by cycloheximide. Insoluble fibronectin deposited by keratinocytes correlated with phagokinetic tracks on particulate gold salts, and added fibronectin, as well as type I collagen and type IV collagen, enhanced motility of keratinocytes. These studies show that production of fibronectin and responsiveness to it are similar in fibroblasts and keratinocytes and demonstrate that fibronectin can act as a matrix factor for keratinocytes.

Production of soluble and cell-associated fibronectin by cultured keratinocytes.

Fibronectin has been demonstrated in epithelial cell types in culture, but published studies of keratinocytes have shown patterns of fibronectin produced by cells grown in medium with serum, which contains fibronectin. Since plasma fibronectin can bind to cells in vitro, cells grown in serum-supplemented media could show artifactual patterns of cell-associated fibronectin. To study insoluble fibronectin produced by keratinocytes, we plated cells in the absence of feeder layers in medium lacking fibronectin. Medium conditioned by metabolically labeled keratinocytes was studied by immunoprecipitation and by extraction with gelatin-Sepharose. Cells grown in fibronectin-free medium were labeled using affinity-purified anti-fibronectin antibody and fluorescein-conjugated antirabbit IgG. Keratinocytes produced soluble fibronectin, since both immunoprecipitation and adsorption to gelatin-Sepharose detected 35S-methionine-labeled material which comigrated with human plasma fibronectin on sodium dodecyl sulfate polyacrylamide gels. Demonstration of insoluble, cell-associated fibronectin was enhanced in Triton X-100-extracted cells and was seen in subcellular fibrillar arrays at both physiologic and reduced Ca++ concentrations, but in intracellular locations only at physiologic Ca++ concentrations. When cells grown in 1.1 mM Ca++ were removed with Triton X-100, diffusely distributed fibrillar fibronectin remained on the surface of the coverslip. Asymmetric "tracks" of fibronectin left by sparsely plated cells suggested movement. Fibronectin is deposited by keratinocytes on the culture surface and may be modulated by culture conditions.

Modulation of the epidermal growth factor receptor of human keratinocytes by calcium ion.

Human keratinocytes grown in medium containing reduced calcium concentrations (0.07 mM) have been found to show altered morphology and decreased differentiation in comparison with cells grown in medium with physiologic calcium concentrations (1-2 mM). Since such alterations could be mediated by growth factors, we measured binding of [125I]epidermal growth factor (EGF). Neonatal keratinocytes were subcultured without feeder layers and grown to confluence in 1.1 mM calcium. Medium was changed and cells incubated for various periods in reduced calcium prior to binding assays. Scatchard plots of binding data showed a 5-fold increase in receptor number with no change in affinity (3 X 10(-9) M) after 4-24 h at 37 degrees C. Maximal binding occurred at 0.02-0.04 mM calcium and decreased sharply with increasing calcium concentrations. The increase could be prevented by calcium added soon after binding began to increase but was altered less after substantial elevation had occurred, although morphologic changes at reduced calcium concentrations were reversed within several hours. Substantial increases in binding of [125I]somatomedin C and [125I]concanavalin A were detected, but binding of [125I]pindalol, a beta-receptor ligand, was changed little. Keratinocytes at reduced calcium concentrations responded to added EGF by decreasing surface EGF receptors briskly in a time- and temperature-dependent fashion. The data suggest that keratinocytes show enhanced binding of EGF and some other cell surface ligands under conditions in which differentiation is retarded.

Unicompartmental arthroplasty of the knee: a review of three-five year follow-up.

Unicompartmental arthroplasty for the treatment of osteoarthritis, that primarily involves a single femorotibial compartment of the knee, has been a controversial procedure. The purpose of this study was to retrospectively review the results of 59 cases in 55 patients, an average of four years after operation. Forty-five of the knees were rated excellent or good and there were eight failures. The thin 6 mm tibial component seemed to be associated with an increased occurrence of loosening and failure of the procedure. There were no failures when thick tibial components were used. The results of this study are encouraging for the use of unicompartmental arthroplasty in the treatment of osteoarthritis of the knee.