Integrative and comparative genomic analyses identify clinically relevant pulmonary carcinoid groups and unveil the supra-carcinoids.

The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary carcinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low- and high-grade lung neuroendocrine neoplasms.

Recognition of tumor cells by immuno-SERS-markers in a microfluidic chip at continuous flow.

SERS active nanoparticles were labeled with a reporter molecule and conjugated with anti-EpCAM antibodies. These immuno SERS markers were mixed with leukocytes, MCF-7 breast cancer cells and polystyrene beads, and the mixture was injected into a microfluidic quartz chip. Raman spectra were acquired at 785 nm excitation with 25 milliseconds exposure time in a continuous flow regime. Spectral unmixing by N-FINDR identified spectral signatures of SERS-labelled cells and polystyrene beads. This approach demonstrated rapid and reproducible SERS-assisted cell detection. Strategies are discussed to further increase the throughput for cell sorting.

Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells.

Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles' shape, material, size, and coating.

Preparation of Core-Shell Hybrid Materials by Producing a Protein Corona Around Magnetic Nanoparticles.

Nanoparticles experience increasing interest for a variety of medical and pharmaceutical applications. When exposing nanomaterials, e.g., magnetic iron oxide nanoparticles (MNP), to human blood, a protein corona consisting of various components is formed immediately. The composition of the corona as well as its amount bound to the particle surface is dependent on different factors, e.g., particle size and surface charge. The actual composition of the formed protein corona might be of major importance for cellular uptake of magnetic nanoparticles. The aim of the present study was to analyze the formation of the protein corona during in vitro serum incubation in dependency of incubation time and temperature. For this, MNP with different shells were incubated in fetal calf serum (FCS, serving as protein source) within a water bath for a defined time and at a defined temperature. Before and after incubation the particles were characterized by a variety of methods. It was found that immediately (seconds) after contact of MNP and FCS, a protein corona is formed on the surface of MNP. This formation led to an increase of particle size and a slight agglomeration of the particles, which was relatively constant during the first minutes of incubation. A longer incubation (from hours to days) resulted in a stronger agglomeration of the FCS incubated MNP. Quantitative analysis (gel electrophoresis) of serum-incubated particles revealed a relatively constant amount of bound proteins during the first minutes of serum incubation. After a longer incubation (>20 min), a considerably higher amount of surface proteins was determined for incubation temperatures below 40 °C. For incubation temperatures above 50 °C, the influence of time was less significant which might be attributed to denaturation of proteins during incubation. Overall, analysis of the molecular weight distribution of proteins found in the corona revealed a clear influence of incubation time and temperature on corona composition.

Towards detection and identification of circulating tumour cells using Raman spectroscopy.

Body fluids are easily accessible and contain valuable indices for medical diagnosis. Fascinating tools are tumour cells circulating in the peripheral blood of cancer patients. As these cells are extremely rare, they constitute a challenge for clinical diagnostics. In this contribution we present the Raman spectroscopic-based identification of different single cells in suspension that are found in peripheral blood of cancer patients including healthy cells like leukocytes and erythrocytes, and tumour cells like leukaemic cells and cells originating from solid tumours. Leukocytes and erythrocytes were isolated from the peripheral blood of healthy donors while myeloid leukaemia cells (OCI-AML3) and breast carcinoma derived cells (MCF-7, BT-20) were obtained from cell cultures. A laser emitting 785 nm light was used for optical trapping the single cells in the laser focus and to excite the Raman spectrum. Support vector machines were applied to develop a supervised classification model with spectra of 1210 cells originating from three different donors and three independent cultivation batches. Distinguishing tumour cells from healthy cells was achieved with a sensitivity of >99.7% and a specificity of >99.5%. In addition, the correct cell types were predicted with an accuracy of approximately 92%.

Rapid screening and sensitive detection of NPM1 (nucleophosmin) exon 12 mutations in acute myeloid leukaemia.

Nucleophosmin mutations of exon 12 (NPM1 mutations) represent the most frequent molecular aberration that can be found in patients with acute myeloid leukaemia (AML) and can be detected in about 35% of AML patients. NPM1 mutations are characterised by four basepair insertions within the region corresponding to the C-terminus of the protein leading to its translocation out of the nucleus. Until now, more than 40 different subsets of mutations have been identified and about 90% of NPM1 mutations are represented by subtype A and B (78% versus 12%, respectively). So far, standard screening of NPM1 mutations using conventional polymerase chain reaction (PCR) followed by capillary electrophoresis is rather time consuming. We established a new method for rapid screening of NPM1 mutations using the fluorescence resonance energy transfer (FRET) principle. Furthermore, based on individual NPM1 mutations type A and B, we designed mutation specific primers to perform a highly sensitive PCR assay that can be applied for the detection of minimal residual disease (MRD). In summary, we demonstrate new methodological approaches for rapid screening of NPM1 mutations as well as for MRD analyses based on the most frequent NPM1 mutations.

ATRA can enhance apoptosis that is induced by Flt3 tyrosine kinase inhibition in Flt3-ITD positive cells.

Among activating Flt3 mutations that have been shown in 25-30% of acute myeloid leukaemia (AML) Flt3-internal tandem duplication (ITD) mutations are predominant. We investigated the influence of all-trans-retinoic acid (ATRA) and granulocyte colony stimulating factor (G-CSF) for their effects on differentiation and apoptosis in human cell lines with different Flt3 variants (THP-1 versus MV4-11 and MOLM13) dependent on the inhibition of Flt3 tyrosine kinase by the bis(lH-2-indolyl)methanone D-65476. While myeloid differentiation was not observed in both Flt3-ITD cell lines (MV4-11 and MOLM13), we demonstrate an enhanced proapoptotic effect of D-65476 in the presence of ATRA that was restricted to the Flt3-ITD expressing cells. The combined treatment with ATRA and D-65476 also led to a pronounced down-regulation of surviv in on mRNA and protein level in Flt3-ITD but not in Flt3 wildtype expressing cells (THP-1). Surprisingly, there was no differential expression of important proteins like Bcl-X(L), Bcl-2 or Bax that might explain enhanced apoptosis. Furthermore, Akt phosphorylation after stimulation with Flt3 ligand dependent on D-65476 was not affected by pretreatment with ATRA. We suggest that regulation of inhibitors of apoptosis might play a crucial role how ATRA can increase the proapoptotic effect of Flt3 inhibitors in myeloid leukemia cells expressing Flt3-ITD. This effect can potentially be exploited for the treatment of Flt3-ITD positive acute myeloid leukemia.

Differential interaction of magnetic nanoparticles with tumor cells and peripheral blood cells.

PURPOSE: The separation of tumor cells from healthy cells is a vital problem in oncology and hematology, especially from peripheral blood. Magnetic assisted cell sorting (MACS) is a possibility to fulfill these needs. METHODS: Tumor cell lines and leukocytes from peripheral blood were incubated with carboxymethyl dextran-coated magnetic nanoparticles under various conditions and separated by MACS. RESULTS: We studied the interaction of magnetic nanoparticles devoid of antibodies with healthy and tumor cells. The magnetic nanoparticles interact with tumor cells and leukocytes and are located predominantly within the cell cytoplasm. Incubation of cell culture cells with magnetic nanoparticles led to a labeling of these cells without reduced biological properties for at least 14 days. The interaction of the magnetic nanoparticles with cells depends on several factors. The ionic strength (osmolality) of the solvent plays an important role. We could show that an increase in osmolality led to a dramatic reduction of labeled leukocytes. Tumor cells, however, are mildly affected. This could be detected not only in pure cultures of tumor cells or leukocytes but also in mixed cell populations. CONCLUSION: This observation gives us the opportunity to selectively label and separate tumor cells but not leukocytes from the peripheral blood.

Molecular characterization of breast cancer cell lines by expression profiling.

PURPOSE: Gene expression patterns provide detailed insights into cellular regulation that reflect minor differences of cellular capacity not accessible by standard descriptions of the cellular phenotype or origin. METHODS: To identify fundamental differences and similarities we analyzed the gene expression patterns of four breast cancer cell lines: MCF-7, SK-BR-3, T-47D, and BT-474. RESULTS: Although only a small subset of genes (597) is represented on the Atlas-cDNA-Array (Clontech) used, clear differences in the expression of a number of genes could be detected. For example, unique high levels of expressions were found for the HLH-protein ID-1 (MCF-7) and the receptor tyrosine kinase erbB2 (SK-BR-3 and T-47D). Most genes analyzed were expressed at comparable levels in all cell lines studied. CONCLUSIONS: For interpretation of the results sets of genes that show similar variation of expression among the cells were grouped together. Furthermore, our analysis allows the assignment of similarity values that lead to a relation profile of the cell lines. How these results correlate with known biological properties of the cell lines is discussed. Additionally, we demonstrate that results obtained by cDNA-Array hybridization for expression of the ErbB receptor family correlate well with competitive RT-PCR, thus confirming the reliability of the cDNA-Array analysis.

Circadian variation of dihydropyrimidine dehydrogenase mRNA expression in leukocytes and serum cortisol levels in patients with advanced gastrointestinal carcinomas compared to healthy controls.

PURPOSE: The activity of dihydropyrimidine dehydrogenase (DPD) - the rate-limiting enzyme in fluorouracil (5-FU) catabolism - has been reported to vary according to the time of day. On the basis of this data, so-called chronomodulated chemotherapy regimens with variable-rate infusions of 5-FU have been investigated in the treatment of advanced colorectal cancer. Recent results suggest lower toxicity of 5-FU by chronomodulated application. However, the pattern of circadian DPD activity levels have been shown to vary considerably. METHODS: We, therefore, studied the circadian changes in mRNA expression of DPD in leukocytes of ten patients with advanced gastrointestinal carcinomas prior to chronomodulated 5-FU-based salvage therapy and in 5five healthy controls. Simultaneously, we measured serum cortisol levels (SCL) to evaluate the endogenous circadian hormone rhythm. RESULTS: SCL displayed a consistent circadian rhythm with the mean peak value of serum cortisol at 8 a.m. and the mean trough value at 11 p.m. both in patients and in controls. However, mean minimum-maximum serum cortisol differences of SCL were significantly lower in patients compared to controls. In the 5fivehealthy controls, a trend towards a circadian rhythm of DPD mRNA expression was observed with the peak of expression at 5 a.m. which was significantly different from the trough at 2 p.m. ( P<0.005 Mann-Whitney-Wilcoxon test). When each control was studied separately, only two individuals showed circadian variations that could be fitted to a cosine wave ( P=0.001, P=0.014, Cosinor analysis). In contrast, DPD mRNA expression in patients with advanced gastrointestinal carcinomas did not demonstrate any consistent circadian rhythm. Pairwise comparisons of groups of DPD mRNA levels at different times of the day did not show significant differences. CONCLUSIONS: In conclusion, our analysis of DPD mRNA expression in leukocytes from healthy controls demonstrates first evidence for a circadian DPD mRNA expression periodicity. In patients with advanced gastrointestinal carcinomas, however, this rhythm seems to be disturbed although circadian endogenous cortisol secretion pattern is maintained.

Molecular cytogenetic characterization of an acquired minute supernumerary marker chromosome as the sole abnormality in a case clinically diagnosed as atypical Philadelphia-negative chronic myelogenous leukaemia.

A case of chronic myelogenous leukaemia (CML) in a 48-year-old man is reported. To the best of our knowledge, this is the first report of a Philadelphia-negative CML with an acquired small supernumerary marker chromosome (SMC) 11 as the sole abnormality. The derivative chromosome 11 was studied in detail using molecular cytogenetic methods; fluorescence in situ hybridization (FISH) using centromere- and region-specific probes for chromosome 11, microdissection, micro-comparative genomic hybridization (micro-CGH) and the recently developed multicolour banding (MCB) technique. The acquired SMC was determined to be a ring chromosome that can be described as r(11)(:p11.2-->q13.1:q14:).

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Bone morphogenetic protein 2 (BMP-2) induces sequential changes of Id gene expression in the breast cancer cell line MCF-7.

Bone morphogenetic proteins (BMPs) are involved in the development of various organs including the mammary gland. They are well-regulated and act in a time-, concentration- and cell-type-specific manner. We found that BMP-2 is expressed in primary breast tumor tissue samples and in breast cancer cell lines. Hybridization of labeled cDNA, obtained from the breast cancer cell line MCF-7, against the Atlas human cDNA expression array revealed differential gene expression depending on BMP-2 treatment. The most prominent changes were observed for the helix-loop-helix proteins Id-1, Id-2 and Id-3. Id-1 expression had increased severalfold after 4 h and was even higher after 24 h. Id-2 and Id-3 were more strongly induced after 4 h and showed no further significant change after 24 h. Analysis of cell-cycle distribution revealed a marked increase of the sub-G1 phase after 48 h in serum-deprived cells. In the presence of BMP-2 no change was observed over 48 h indicating that BMP-2 does not induce apoptosis. In addition, expression of caspase-3 was reduced in BMP-2-treated cells after 24 h. In summary, our results clearly indicate that BMP-2 is a susceptibility factor keeping the cells ready for the integration of various other signals for cell progression.

Expression, regulation and clinical significance of bone morphogenetic protein 6 in esophageal squamous-cell carcinoma.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines. BMP-6 is involved in early steps of keratinogenesis. Esophageal squamous-cell carcinoma often shows abnormal keratinization. The aim of this study was to examine the expression and regulation of BMP-6 in esophageal carcinoma and to determine the relationship of its expression with tumor characteristics and survival. Avidin-biotin-peroxidase staining was performed using paraffin-wax-embedded tumor specimens collected from 172 patients with esophageal squamous-cell carcinoma, who underwent potentially curative surgery. Expression of BMP-6 and EGF-receptor in the esophageal-squamous-cell-carcinoma cell line COLO-680N and the hypopharyngeal-squamous-cell-carcinoma cell line FaDu was assayed with specific RT-PCR. In standard cell-cultivation conditions, COLO-680N and FaDu cells showed distinct expression of BMP-6, whereas in serum-free conditions BMP-6 expression was hardly detectable. The addition of EGF or EGF-related growth factors could elevate BMP-6 mRNA content. Immunohistochemically, all 172 tumors exhibited cytoplasmic immunoreactivity for BMP-6. The content of BMP-6 protein was inversely correlated with tumor differentiation (p < 0.01). Univariate survival analysis revealed that 74 patients with tumors of low immunoreactivity had higher 2-year and 5-year survival rates (50.0%/27.7%) than 75 patients with tumors of high immunoreactive scores (33.3%/21.6%). (p < 0.05). We conclude that BMP-6 is expressed in esophageal squamous-cell carcinoma. EGF-receptor activation is crucial for regulation of BMP-6 expression. BMP-6 protein content is correlated with the grade of tumor-cell differentiation and may add to indications of poor prognosis.

Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor.

Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP-6 is involved in numerous developmental processes. We have demonstrated expression of BMP-6 in breast cancer cell lines by RT-PCR and immuno-histochemistry. The level of BMP-6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP-6 mRNA levels in a dose-dependent manner, with a maximum at 50 ng/ml EGF under serum-free conditions in hormone-sensitive (MCF-7) and in hormone-insensitive (SK-BR-3) breast cancer cell lines. The EGF-like growth factors transforming growth factor-alpha, amphiregulin and betacellulin were also able to elevate the BMP-6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG 1517 repressed the inductive effect of these growth factors, indicating an EGF receptor-mediated regulation of BMP-6 mRNA. In addition, BMP-6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor-free resection margins. In 12 of these 18 patients, at least a 10-fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor-free samples was observed. This suggests a putative relationship between EGF receptor and BMP-6 mRNA levels in breast cancer.

Antagonistic actions of activin A and BMP-2/4 control dorsal lip-specific activation of the early response gene XFD-1' in Xenopus laevis embryos.

Transcription of the early response gene XFD-1' (XFKH1) in the dorsal lip (Spemann organizer) of Xenopus embryos is activated by dorsal mesoderm inducing factors. Promoter studies revealed the presence of an activin A response element (ARE) which is both necessary and sufficient for transcriptional activation of reporter genes in animal cap explants incubated with activin A. Surprisingly, this ARE is also active within vegetal explants in the absence of exogenously added inducers, but an additional inhibitory response element prevents transcription of the XFD-1' gene in the ventral/vegetal region of the embryo in vivo. This element is located upstream of the ARE, it responds to bone morphogenic proteins 2 and 4 (BMP-2/4) triggered signals and it overrides the activating properties of the ARE. Expression patterns of BMP-2 and BMP-4 in the late blastula stage embryo and, especially, their absence from the dorsal blastopore lip may thus control the spatial transcription of the XFD-1' gene. Accordingly, the temporal activation and the spatial restriction of XFD-1' gene activity to the Spemann organizer is regulated by antagonistic actions of two distinct members of the TGF-beta family (activin and BMP) which act on different promoter elements.

A fork head related multigene family is transcribed in Xenopus laevis embryos.

We have isolated and sequenced ten different members of the fork head/HNF-3 multigene family from Xenopus laevis which have been termed Xenopus fork head domain related (XFD) genes 1 to 10. Another four isolated genes (XFD' genes) represent pseudo-allelic variants which arose by an ancient tetraploidization within this species. Whereas all genes of this multigene family exhibit a high degree of sequence homology within the evolutionary conserved fork head domain, sequences outside this module are substantially different. Based upon sequence homologies over the entire coding sequences, XFD-7/7' represent the Xenopus homologs to the rodent hepatocyte nuclear factor HNF-3 alpha, while XFD-3/3' encode the homologs to HNF-3 beta. Here we present an analysis of the temporal transcription pattern of XFD genes 1 to 10 during embryogenesis and in some adult tissues. Eight of these XFD genes are activated during embryonic development, but show different and distinct transcription profiles. The localization of transcripts was determined by whole-mount in situ hybridization. Although transcription of individual XFD genes partially overlaps, each gene is characterized by means of a specific spatial pattern of transcriptional activity.

Bone morphogenetic protein 2 in the early development of Xenopus laevis.

The temporal and spatial transcription patterns of the Xenopus laevis Bone morphogenetic protein 2 (BMP-2) gene have been investigated. Unlike the closely related BMP-4 gene, the BMP-2 gene is strongly transcribed during oogenesis. Besides some enrichment within the animal half, maternal BMP-2 transcripts are ubiquitously distributed in the early cleavage stage embryos but rapidly decline during gastrulation. Zygotic transcription of this gene starts during early neurulation and transcripts are subsequently localized to neural crest cells, olfactory placodes, pineal body and heart anlage. Microinjection of BMP-2 RNA into the two dorsal blastomeres of 4-cell stage embryos leads to ventralization of developing embryos. This coincides with a decrease of transcripts from dorsal marker genes (beta-tubulin, alpha-actin) but not from ventral marker genes (alpha-globin). BMP-2 overexpression inhibits transcription of the early response gene XFD-1, a fork head/HNF-3 related transcription factor expressed in the dorsal lip, but stimulates transcription of the posterior/ventral marker gene Xhox3, a member of the helix-turn-helix family. Activin A incubated animal caps from BMP-2 RNA injected embryos show transcription of ventral but an inhibition of dorsal marker genes; thus, BMP-2 overrides the dorsalizing activity of activin A. The results demonstrate that BMP-2 overexpression exerts very similar effects as have previously been described for BMP-4, and they suggest that BMP-2 may act already as a maternal factor in ventral mesoderm formation.

Spatial and temporal transcription patterns of the forkhead related XFD-2/XFD-2' genes in Xenopus laevis embryos.

Two novel fork head related cDNA sequences, termed XFD-2 and XFD-2', have been isolated from a Xenopus laevis gastrula stage cDNA library. XFD-2 and XFD-2' proteins share 88% sequence identity; a comparison of their fork head domains yields 96% identity. Such close homology suggests that the two genes represent pseudo-allelic variants of a common ancestor and probably arose by the ancient tetraploidization event in this species. Both genes are activated at midblastula transition. Main transcriptional activity is found during blastula and gastrula stages of development; thereafter, there is a gradual decrease of transcripts until somite segregation stages. Whole mount in situ hybridisation of blastula stage embryos reveals that the genes are initially transcribed within the animal hemisphere. Subsequently, we observe their transcription in a circumferential mode along the marginal zone, i.e., within the forming mesoderm. During gastrulation, these cells enter the blastoporus at the ventral, lateral and dorsal sites. At the end of gastrula and during neural stages transcripts are localized within somitogenic mesoderm, notochord, lateral and ventral mesoderm, neural floor plate, spinal cords and in the developing brain.