Thomas Armbrust

Cytokines in the liver.

Cytokines comprise a group of small proteins released from cells in order to influence the function of other cells. By binding to highly specific cell-surface receptors, they trigger a vast array of intracellular signalling cascades. Cytokines have been described as interleukins, growth factors, interferons and chemokines. Unlike hormones, which act in a similar way, cytokines are produced by many different types of cell and act on many other types. Most of them are produced only after certain stimuli. The most intense field of cytokine activity is without doubt host defence. The liver resembles a central organ of cytokine activity due to the fact that it hosts hepatocytes, which are highly susceptible to the activity of cytokines in a variety of physiological and pathophysiological processes. Moreover, the non-parenchymal cells of the liver, in particular Kupffer cells (KCs), the resident tissue macrophages of the liver, are able to synthesize a variety of cytokines that may act systemically on any other organ of the body, or in a paracrine manner on hepatocytes and other non-parenchymal liver cells. A classic example of how cytokines act can be observed during the acute phase reaction discussed in this article. The role of cytokines in liver development, acute liver injury, liver regeneration, liver fibrosis and liver metastasis is also discussed.

Mast cells distribution in human liver disease and experimental rat liver fibrosis. Indications for mast cell participation in development of liver fibrosis

BACKGROUND/AIMS The development of liver fibrosis due to chronic liver diseases is thought to be mediated by inflammatory cells releasing fibrogenic mediators that activate fat-storing cells (Ito-cells). Recently, the involvement of mast cells in fibrogenesis has been suggested. We studied the distribution of these cells in normal human liver and human nonfibrotic and fibrotic liver disease as well as in normal rat liver and acutely and chronically injured rat liver (CCl4 model). METHODS Mast cells were identified by histochemical and immunohistochemical methods. The immunoreactivity of liver and comparatively of rat peritoneal mast cells to the serpins alpha1-antitrypsin, alpha1-antichymotrypsin and antithrombin III was also studied. RESULTS In normal human and rat liver, mast cells were rarely found in portal tracts, and there was no change in cell numbers in nonfibrotic human or acutely injured rat livers. In contrast, cirrhotic human and rat livers contained numerous mast cells in the portal tracts and the fibrous septa. They exhibited strong immunoreactivity to the serpins, as did rat peritoneal mast cells. CONCLUSIONS The results indicate that in the late stages of liver fibrogenesis, mast cells may be involved by displaying protease inhibitory activity in the fibrotic septa.

An oncogenetic tree model in gastrointestinal stromal tumours (GISTs) identifies different pathways of cytogenetic evolution with prognostic implications

To model the cytogenetic evolution in gastrointestinal stromal tumour (GIST), an oncogenetic tree model was reconstructed using comparative genomic hybridization data from 203 primary GISTs (116 gastric and 87 intestinal GISTs, including 151 newly analysed cases), with follow‐up available in 173 cases (mean 40 months; maximum 133 months). The oncogenetic tree model identified three major cytogenetic pathways: one initiated by − 14q, one by − 1p, and another by − 22q. The − 14q pathway mainly characterized gastric tumours with predominantly stable karyotypes and more favourable clinical course. On the other hand, the − 1p pathway was more characteristic of intestinal GISTs, with an increased capacity for cytogenetic complexity and more aggressive clinical course. Loss of 22q, more closely associated with − 1p than − 14q, appeared to initiate the critical transition to an unfavourable cytogenetic subpathway. This − 22q pathway included accumulation of + 8q, − 9p, and − 9q, which could all predict disease‐free survival in addition to tumour site. Thus, insights into the cytogenetic evolution obtained from oncogenetic tree models may eventually help to gain a better understanding of the heterogeneous site‐dependent biological behaviour of GISTs. Copyright

Functional characterization of two different Kupffer cell populations of normal rat liver

BACKGROUND Kupffer cells of the liver represent the largest population of tissue macrophages. Small and large Kupffer cells were distinguished in normal liver, leading to the suggestion that they have different functions. This study intends to further characterize small and large Kupffer cells of normal rat liver in vivo and in vitro. METHODS Sections of rat liver were investigated by double-staining immunofluorescence with the monoclonal antibodies ED1 and ED2. Isolated nonparenchymal liver cells were separated according to size to obtain small and large Kupffer cells. In culture, phagocytosis was studied by zymosan ingestion and cell proliferation by incorporation of 3H-thymidine. Synthesis of the proteins C1-inhibitor, apolipoprotein E and interleukin-1 was studied by endogenous labeling of newly synthesized proteins, immunoprecipitation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. RESULTS ED1+ ED2+ Kupffer cells were located in the liver along the sinusoids. ED1+ ED2+ cells were found mainly located around the central vein and portal vessels. By counterflow elution, small ED1+ ED2- cells were separated from larger ED1+ ED2+ cells and cultured. The larger cells abundantly synthesized C1-inhibitor and apolipoprotein E, while the small cells synthesized only trace amounts of these proteins. Interferon-gamma increased C1-inhibitor synthesis in small (5-fold) and large cells (1.5-fold). 3H-thymidine incorporation was 11-fold higher in small than in large cells. However, lipopolysaccharide-induced pro-interleukin-1 alpha and pro-interleukin-1 beta synthesis and phagocytic activity were similar in both populations. CONCLUSIONS The data demonstrate two different populations of mononuclear phagocytes in normal rat liver well distinguished by immunocytochemical and functional markers.

Multicentric sporadic gastrointestinal stromal tumors (GISTs) of the stomach with distinct clonal origin: differential diagnosis to familial and syndromal GIST variants and peritoneal metastasis.

Most sporadic gastrointestinal stromal tumors (GISTs) occur solitary, whereas a multicentric appearance is suspicious for a familial or syndromal setting such as with germline mutations of proto-oncogene tyrosine protein kinase Kit (KIT) or platelet derived growth factor receptor alpha (PDGFRA), or even for metastases. The aim of this study was to evaluate whether multicentric sporadic GISTs are of clonal origin. Four patients with 1 clinically apparent tumor (mean size 5.6 cm) and 1 to 3 further small incidental tumors (mean size 0.7 cm) were analysed by mutation analysis and comparative genomic hybridization for mutations of KIT and PDGFRA and chromosomal imbalances in their tumors. No clinicopathologic features have been found being indicative of one of the established familial or syndromal GIST variants. Each of the small GISTs were localized in the muscularis propria, and were visible from the serosal but not from the mucosal side. Different mutations of KIT and PDGFRA were present among individual tumors of each patient, and germline mutation of KIT and PDGFRA could be excluded. Comparative genomic hybridization revealed a mean count of 7 chromosomal imbalances in the clinically apparent tumors compared with a mean count of 0.3 in the small incidental counterparts. Sporadic GISTs can appear multicentric by coincidence. They are an important differential diagnosis to familial and syndromal GIST variants, or even to peritoneal metastases. Different mutations of KIT and PDGFRA among individual tumors in 1 patient refer to different clonal origin of multicentric sporadic GISTs. The type of mutation of KIT and PDGFRA was independent of tumor size, whereas small GISTs <1 cm rarely had genomic imbalances.

Successful treatment of hepatocellular carcinoma with the tyrosine kinase inhibitor imatinib in a patient with liver cirrhosis

Several mechanisms of development of hepatocellular carcinoma (HCC) in patients with liver cirrhosis have been discussed. One hypothesis suggests that the somatic stem cells of the liver, the so-called oval cells, may undergo malignant transformation. Oval cells are derived from the biliary cells of the canal of Hering and are characterized by c-kit-positivity, the transmembrane receptor of stem cell factor. Constitutively activated tyrosine kinases have been identified as major pathogenetic mechanisms in the development of malignant diseases like gastrointestinal stromal tumors (c-kit) and chronic myelogenous leukemia (bcr–abl). The prognosis of these diseases improved enormously since the drug imatinib, a tyrosine kinase inhibitor of c-kit and bcr–abl, was introduced. Here we report the successful cure of a patient with liver cancer by this tyrosine kinase inhibitor.

Site-dependent differential KIT and PDGFRA expression in gastric and intestinal gastrointestinal stromal tumors.

In gastrointestinal stromal tumors (GISTs), mutually exclusive gain-of-function mutations of KIT and PDGFRA are associated with different mutation-dependent clinical behavior. Taking into account the well-known different clinical behavior of GISTs from the stomach or the intestine, the aim of the current study is to evaluate the mutation- and site-dependent effects on mRNA and protein expression of KIT and PDGFRA in a large series of primary GISTs. Fresh-frozen tissue of 53 primary GISTs from gastric (75%) or intestinal (25%) sites were analyzed for mutation of KIT or PDGFRA using direct sequencing. Furthermore, KIT and PDGFRA mRNA and protein expression were determined using quantitative RT–PCR and quantitative densitometric evaluation of Western blot data. Each tumor either had a mutation of KIT (79%) or PDGFRA (21%). All GISTs with PDGFRA mutation were from gastric sites. Mutation-dependently, GISTs with KIT mutation had a significantly higher expression of KIT and at the same time a significantly lower expression of PDGFRA compared to GISTs with PDGFRA mutation. Site-dependently, gastric GISTs had a significantly higher expression of PDGFRA and a significantly lower expression of KIT compared to intestinal GISTs. Additionally, even if the KIT-mutated GISTs alone were considered, a significantly higher expression of PDGFRA could be observed in gastric than in intestinal tumors. We also found a significant correlation between a higher protein expression of PDGFRA and longer disease-free survival. The correlation of gastric site and PDGFRA mutation with higher PDGFRA expression and longer disease-free survival suggests different regulatory roles of KIT and PDGFRA gene expression on the control of cell proliferation, and, thereby on clinical behavior. The higher PDGFRA expression in gastric GISTs possibly contributes to the well-known site-dependent clinical behavior.

Immune cells in primary gastrointestinal stromal tumors

Introduction Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. They are regarded as having relatively uniform histology, although their potential for malignant behavior varies. Despite a strong promoting role of tumor-infiltrating innate immune cells in neoplastic progression, the presence of immune cells in GISTs has not yet been studied. Methods A total of 47 untreated, c-kit-positive primary GISTs were immunohistochemically analyzed to distinguish histiocytic and dendritic cells (DCs) (KIM-1P, fascin, and CD68) from cells of lymphoplasmacellular origin (CD3, CD20, and CD56). Furthermore, the gene expression of proinflammatory cytokines was characterized by real-time, reverse transcription-PCR analysis of total RNA extracted from frozen tissue samples. Results KIM-1P+ cells were the dominant immune cells (851±295 cells/mm2) and were scattered among the tumor cells. Most of the KIM-1P+ cells showed cellular projections characteristic of DCs. Fascin positivity identified a subgroup of DCs. In comparison to KIM-1P+ cells, there were significantly fewer CD68+ macrophages (196±217 cells/mm2). CD3+ T cells were the dominant lymphocytes (201±331 cells/mm2), whereas B cells (60±126 cells/mm2) were few. On transcriptional level, a concomitant gene expression of cytokines for the classical acute phase cytokines TNF-&agr; and IL-6 was missing, thus supporting the rather innate status of immune cells. Conclusion GISTs contain, beside T lymphocytes, a high number of monocyte-derived cells, which we suggest are, at least in part, immature DCs. Together with the lack of gene expression of inflammatory cytokines in tumor tissue our results point to a possible ‘symbiotic relationship’ between the tumor and the local immune cells.

Increased KIT signalling with up-regulation of cyclin D correlates to accelerated proliferation and shorter disease-free survival in gastrointestinal stromal tumours (GISTs) with KIT exon 11 deletions†

Gastrointestinal stromal tumours (GISTs) with deletions in KIT exon 11 are characterized by higher proliferation rates and shorter disease‐free survival times, compared to GISTs with KIT exon 11 point mutations. Up‐regulation of cyclin D is a crucial event for entry into the G1 phase of the cell cycle, and links mitogenic signalling to cell proliferation. Signalling from activated KIT to cyclin D is directed through the RAS/RAF/ERK, PI3K/AKT/mTOR/EIF4E, and JAK/STATs cascades. ERK and STATs initiate mRNA transcription of cyclin D, whereas EIF4E activation leads to increased translation efficiency and reduced degradation of cyclin D protein. The aim of the current study was to analyse the mRNA and protein expression as well as protein phosphorylation of central hubs of these signalling cascades in primary GISTs, to evaluate whether tumours with KIT exon 11 deletions and point mutations differently utilize these pathways. GISTs with KIT exon 11 deletions had significantly higher mitotic counts, higher proliferation rates, and shorter disease‐free survival times. In line with this, they had significantly higher expression of cyclin D on the mRNA and protein level. Furthermore, there was a significantly higher amount of phosphorylated ERK1/2, and a higher protein amount of STAT3, mTOR, and EIF4E. PI3K and phosphorylated AKT were also up‐regulated, but this was not significant. Ultimately, GISTs with KIT exon 11 deletions had significantly higher phosphorylation of the central negative cell‐cycle regulator RB. Phosphorylation of RB is accomplished by activated cyclin D/CDK4/6 complex, and marks a central event in the release of the cell cycle. Altogether, these observations suggest increased KIT signalling with up‐regulation of cyclin D as the basis for the unfavourable clinical course in GISTs with KIT exon 11 deletions. Copyright

Use of total and unbound imatinib and metabolite LC-MS/MS assay to understand individual responses in CML and GIST patients.

Objectives: Trough total imatinib (t-IM) concentrations have been reported to be associated with therapeutic and toxic responses in patients with chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST). Little is known about the relationships between effects and concentrations of either unbound imatinib (f-IM) or imatinibs major metabolite, N-desmethyl imatinib (NDI). In part, this is because of the lack of a single, validated, well-described clinically useful assay for these measurements. The authors report the development and application of such an assay. Materials and Methods: A single liquid-chromatography tandem-mass-spectrometry assay was used to monitor t-IM, f-IM, and t-NDI concentrations in CML and GIST patients treated at a tertiary German teaching hospital. The assay was also validated for measuring other kinase inhibitors, including t-nilotinib, sunitinib, and erlotinib. Ultrafiltration assays were validated and used to measure f-IM and to compare free fractions to plasma α1-acid glycoprotein concentrations (AGP). Results: The assays were linear over a working range (in micrograms per liter) of 8.4-8370, 8.3-4165, and 1.0-250 and had within- and between-run coefficient of variance of <7%, <12%, and <9% for t-IM, t-NDI, and f-IM, respectively. The f-IM assay was reproducible despite high (25.2%-31.6%) but concentration-independent binding to ultrafiltration devices. Clinically relevant results, such as nondetectable (ND) t-IM (<8.4 μg/L) in non-responders and >1500 μg/L in patients with major toxicity, were found. Of 156 total samples from 68 adult CML patients and 127 total samples from 42 adult GIST, only 48 samples from 22 CML patients and 40 samples from 20 GIST patients were trough samples with adequate dosing and collection information. More than half (27 of 48 CML and 24 of 40 GIST) had t-IM concentrations ≥10% below recommended target concentrations (1002 μg/L for CML and 1100 μg/L for GIST). Concentrations >50% over targets were also found in 6 of 48 CML and 4 of 40 GIST samples. Wide variations in concentrations of t-IM (range, ND to 2973 μg/L), t-NDI (range, ND to 659 μg/L), f-IM (range, 8.3-262 μg/L), and t-IM:f-IM ratios (range, 2.6%-14%) were found both between and within patients. A statistically significant association (Spearman correlation coefficient and P value for all samples, r = 0.290 and P = 0.023; for trough only, r = −0.585 and P = 0.028) was found between AGP and f-IM concentrations but wide interpatient and intrapatient variations made individual predictions unreliable. Conclusions: The liquid-chromatography tandem-mass-spectrometry methods developed provided information useful to understand individual responses to therapy even though necessary sampling and dosing information was often not available. Wide unpredictable variations in t-IM, t-NDI, and f-IM were found. Clinical outcome trials are needed to examine whether f-IM or NDI monitoring can improve the ability to predict individual responses.