Kit-negative fibroblast-like cells expressing SK3, a Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channel,in the gut musculature in health and disease

The apamin-sensitive component of the inhibitory response of the gastrointestinal musculature involves the small conductance Ca(2+)-activated K(+) channel SK3. Kit-immunoreactive (ir) interstitial cells of Cajal appear to be involved in nitrergic inhibition while the role of the recently described CD34-ir fibroblast-like cells adjacent to, but distinct from, the cells of Cajal remains elusive. The distribution of SK3 was studied by immunohistochemistry in the normal human gut, in motility disorders with a lack of cells of Cajal (infantile hypertrophic pyloric stenosis and Hirschsprung's disease) and in mice deficient in cells of Cajal. SK3 immunoreactivity was observed exclusively in Kit-negative interstitial cells adjacent to, but distinct from, the Kit-ir interstitial cells of Cajal in the normal gut. The distribution of SK3-ir cells was not altered in conditions where cells of Cajal were lacking. These cells were CD34-ir fibroblast-like cells in the human gut and in the mouse stomach, while SK3-ir cells in the mouse intestine were CD34 negative. As SK channels are reportedly involved in inhibitory neurotransmission, our morphological observations suggest that SK3-ir interstitial cells, distinct from the Kit-ir interstitial cells of Cajal, may represent a novel cellular component in the control of excitability of the digestive musculature. Further studies will be required to directly address the function of these cells.     

Distribution of the intermediate filament nestin in the muscularis propria of the human gastrointestinal tract

The intermediate filament nestin is expressed in neural stem cells, neuroectodermal tumors and various adult tissues. In the gastrointestinal (GI) tract, nestin has been reported in glial cells. Recently, nestin has been reported in interstitial cells of Cajal (ICC) and in gastrointestinal stromal tumors, thought to derive from ICC. Here we investigated nestin immunoreactivity (-ir) in the normal human GI tract, with emphasis on Kit-ir ICC. Two different antibodies specific for human nestin and multicolor high-resolution confocal microscopy were used on material from our human GI tissue collection. The staining pattern of both nestin antibodies was similar. In labeled cells, nestin-ir appeared filamentous. Most intramuscular ICC in antrum and all myenteric ICC (ICC-MP) in small intestine were nestin-ir, while nestin-ir was not detected in deep muscular plexus ICC. In the colon, some - but not all - ICC-MP and most ICC in the circular musculature were nestin-ir while nestin-ir was not detected in ICC in the longitudinal musculature and in the submuscular plexus. In addition, many Kit-negative cells were nestin-ir in all regions. Neurons and smooth muscle cells were consistently nestin negative, while most S100-ir glial cells were nestin-ir. In addition, nestin-ir was also present in some CD34-ir fibroblast-like cells, in endothelium and in other cell types in the mucosa and serosa. In conclusion, nestin-ir is abundantly present in the normal human GI tract. Among a number of cell types, several, but not all, subpopulations of Kit-ir ICC were nestin-ir. The functional significance of nestin in the GI tract remains obscure.     

Kit K641E oncogene up-regulates Sprouty homolog 4 and Trophoblast glycoprotein in interstitial cells of Cajal in a murine model of gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the receptor tyrosine kinase KIT are present in most GIST. KIT K642E was originally identified in sporadic GIST and later found in the germ line of a familial GIST cohort. A mouse model harbouring a germline Kit K641E mutant was created to model familial GIST. The expression profile was investigated in the gastric antrum of the Kit ^K641E murine GIST model by microarray, quantitative PCR and immunofluorescence. Gja1/Cx43 , Gpc6 , Gpr133 , Pacrg , Pde3a , Prkar2b , Prkcq/Pkce , Rasd2 , Spry4 and Tpbg/5T4 were found to be up-regulated. The proteins encoded by Gja1/Cx43 , Pde3a , Prkcq/Pkce were localized in Kit-ir ICC in wild-type and Kit ^K641E animals while Spry4 and Tpbg/5T4 were detected in Kit-ir cells only in Kit ^K641E, but not in Kit ^WT/WT animals. Most up-regulated genes in this mouse model belong to the gene expression profile of human GIST but also to the profile of normal Kit⁺ ICC in the mouse small intestine. Spry4 and Tpbg/5T4 may represent candidates for targeted therapeutic approaches in GIST with oncogenic KIT mutations.     

Neurotensin receptor 1 is expressed in gastrointestinal stromal tumors but not in interstitial cells of Cajal

Gastrointestinal stromal tumors (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the KIT or PDGFRA receptor tyrosine kinases are present in the majority of GIST, leading to ligand-independent activation of the intracellular signal transduction pathways. We previously investigated the gene expression profile in the murine Kit(K641E) GIST model and identified Ntsr1 mRNA, encoding the Neurotensin receptor 1, amongst the upregulated genes. Here we characterized Ntsr1 mRNA and protein expression in the murine Kit(K641E) GIST model and in tissue microarrays of human GIST. Ntsr1 mRNA upregulation in Kit(K641E) animals was confirmed by quantitative PCR. Ntsr1 immunoreactivity was not detected in the Kit positive ICC of WT mice, but was present in the Kit positive hyperplasia of Kit(K641E) mice. In the normal human gut, NTSR1 immunoreactivity was detected in myenteric neurons but not in KIT positive ICC. Two independent tissue microarrays, including a total of 97 GIST, revealed NTSR1 immunoreactivity in all specimens, including the KIT negative GIST with PDGFRA mutation. NTSR1 immunoreactivity exhibited nuclear, cytoplasmic or mixed patterns, which might relate to variable levels of NTSR1 activation. As studies using radio-labeled NTSR1 ligand analogues for whole body tumor imaging and for targeted therapeutic interventions have already been reported, this study opens new perspectives for similar approaches in GIST.     

Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

Background

Interstitial cells of Cajal (ICC) have been identified in urinary bladder of several species, but their presence in mice remains uncertain. Meanwhile, dozens of reports indicate that dysregulation of connexin 43 plays an important role in bladder overactivity, but its localization has not been clearly defined, with reports of expression in either the smooth muscle or in myofibroblasts. We recently identified a population of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) positive cells that resemble ICC and are distinct from smooth muscle, fibroblasts, myofibroblasts and neurons. Thus we sought to define more clearly the molecular signature of ICC and in doing so resolve some of these uncertainties.

Principle findings

Immunofluorescent localization revealed that NTPDase2-positive cells lie closely adjacent to smooth muscle but are separate from them. NTPDase2 positive cells exhibited co-localization with the widely accepted ICC marker - c-kit. They were further shown to co-localize with other ICC markers CD34 and Ano1, but not with mast cell marker tryptase. Significantly, they show convincing co-localization with connexin 43, which was not present in smooth muscle. The identity of these cells as ICC was further confirmed by the presence of three mesenchymal markers – vimentin, desmin, and PDGFβ receptor, which indicates their mesenchymal origin. Finally, we observed for the first time, the presence of merlin/neurofibromin 2 in ICC. Normally considered a neuronal protein, the presence of merlin suggests ICC in bladder may have a role in neurotransmission.

Conclusions

NTPDase2 positive cells in mice bladder are ICC, which can be defined by the presence of c-Kit, CD34, Ano1, NTPDase2, connexin 43, vimentin, desmin, PDGFβ receptor and merlin/NF2. These data establish a definitive molecular expression profile, which can be used to assist in explorations of their functional roles, and the presence of NTPDase2 suggests that purinergic signaling plays a role in regulation of ICC function.     

Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine

Specific functions of interstitial cells of Cajal (ICC) have been linked to distinct classes that differ by morphology and distribution. In the small intestine, slow wave-generating ICC are located in the myenteric region (ICC-MY), whereas ICC that mediate neuromuscular neurotransmission occur either throughout the circular muscle layer (intramuscular ICC, ICC-IM) or in association with the deep muscular plexus (ICC-DMP). Selective isolation of ICC to characterize specific properties has been difficult. Recently, neurokinin-1 receptors have been detected in murine ICC-DMP and neurons but not in ICC-MY. Here we identified and isolated ICC-DMP/IM by receptor-mediated internalization of fluorescent substance P and Kit immunofluorescence. Specificity of labeling was verified by confocal microscopy. Mouse and human ICC-DMP/IM were detected in suspension by fluorescent microscopy and harvested for RT-PCR with micropipettes. The isolated cells expressed Kit but not markers for neurons, smooth muscle, or antigen-presenting cells. ICC-DMP expressed neurokinin-1 receptor, M(2) and M(3) muscarinic receptors, P2Y(1) and P2Y(4) purinergic receptors, VIP receptor 2, soluble guanylate cyclase-1 subunits, and protein kinase G. L- or T-type Ca(2+) channels were not detected in these cells. ICC-MY and ICC-DMP were simultaneously detected and enumerated by flow cytometry and sorted to purity by fluorescence-activated cell sorting. In summary, functional classes of ICC have distinct molecular identities that can be used to selectively identify and harvest these cells with, for example, receptor-mediated uptake of substance P and Kit immunofluorescence. ICC-DMP express neurotransmitter receptors and signaling intermediate molecules that are consistent with their role in neuromuscular neurotransmission.     

Telocytes express PDGFRα in the human gastrointestinal tract

Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34‐positive/c‐kit‐negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c‐kit‐positive/CD34‐negative/platelet‐derived growth factor receptor α (PDGFRα)‐negative interstitial cells of Cajal (ICC) and the PDGFRα‐positive/c‐kit‐negative fibroblast‐like cells (FLC). As TC display the same features and locations of the PDGFRα‐positive cells, we investigated whether TC and PDGFRα‐positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c‐kit and CD34/c‐kit double immunolabelling was performed in full‐thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34‐positive. TC formed a three‐dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c‐kit‐positive and CD34/PDGFRα‐negative. In conclusion, in the human GI tract the TC are PDGFRα‐positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region‐specific roles.     

Normal and leukemic CD34-negative human hematopoietic stem cells

Considerable progress has been made in recent years in purifying human and murine hematopoietic stem cells. The essential marker identified is the sialomucin CD34, which is expressed on primitive cells and downregulated as they differentiate into more abundant mature cells. CD34 is not unique to stem cells, however, as it is also expressed on clonogenic progenitors and some endothelial cells. Nevertheless, all clinical and experimental protocols are targeted to CD34+ cells enriched by a variety of selection methods. Recent studies in both the murine and human systems have indicated that some stem cells capable of multilineage repopulation do not express detectable levels of cell surface CD34. These studies challenge the dogma that all human repopulating cells are found in the CD34+ subset. However, the precise relationship between CD34- and CD34+ stem cells is still not well understood. In this review, the results on the discovery of the CD34- repopulating cell are summarized and the impacts this discovery may have, both clinically and in our understanding of the organization of the human hematopoietic system, are examined.     

Glial cells,but not interstitial cells,express P2X7, an ionotropic purinergic receptor,in rat gastrointestinal musculature

Purinergic (ATP) neurotransmission is a component of the inhibitory response of the musculature in various regions of the gastrointestinal tract. So far, seven ionotropic purinergic receptors (P2X1-7) have been cloned. As specific antibodies become available, their respective distribution in the gastrointestinal tract can be elucidated. Here, we used high-resolution tricolor confocal microscopy, to study the distribution of P2X7-immunoreactive (-ir) cells in the muscularis propria of the rat stomach, small intestine, and colon. Smooth muscle cells, KIT-ir interstitial cells of Cajal, and CD34/SK3-ir fibroblastlike cells were P2X7-negative, whereas P2X7 immunoreactivity was observed in nerves and S100-ir glial cells. In all regions studied, P2X7 immunoreactivity was also observed in myenteric and submucosal ganglia, where perineuronal nerve endings appeared brightly labeled. Our observations suggest that purinergic signaling could influence the enteric glia through P2X7 receptors.     

Immunomagnetic enrichment of interstitial cells of Cajal

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.     

The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions

The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.     

Ether-a-go-go-related gene 3 is the main candidate for the E-4031-sensitive potassium current in the pacemaker interstitial cells of Cajal

The interstitial cells of Cajal (ICC), as pacemaker cells of the gut, contribute to rhythmic peristalsis and muscle excitability through initiation of slow-wave activity, which subsequently actively propagates into the musculature. An E-4031-sensitive K(+) current makes a critical contribution to membrane potential in ICC. This study provides novel features of this current in ICC in physiological solutions and seeks to identify the channel isoform. In situ hybridization and Kit immunohistochemistry were combined to assess ether-a-go-go-related gene (ERG) mRNA expression in ICC in mouse jejunum, while the translated message was assessed by immunofluorescence colocalization of ERG and Kit proteins. E-4031-sensitive currents in cultured ICC were studied by the whole cell patch-clamp method, with physiological K(+) concentration in the bath and the pipette. In situ hybridization combined with Kit immunohistochemistry detected m-erg1 and m-erg3, but not m-erg2, mRNA in ICC. ERG3 protein was colocalized with Kit-immunoreactive ICC in jejunum sections, but ERG1 protein was visualized only in the smooth muscle cells. At physiological K(+) concentration, the E-4031-sensitive outward current in ICC was different from its counterpart in cardiac and gut smooth muscle cells. In particular, inactivation upon depolarization and recovery from inactivation by hyperpolarization were modest in ICC. In summary, the E-4031-sensitive currents influence the kinetics of the pacemaker activity in ICC and contribute to maintenance of the resting membrane potential in smooth muscle cells, which together constitute a marked effect on tissue excitability. Whereas this current is mediated by ERG1 in smooth muscle, it is primarily mediated by ERG3 in ICC.     

Ultrastructure of Cajal-like interstitial cells in the human detrusor

The aim of this ultrastructural study was to examine the human detrusor for interstitial cells of Cajal (ICC)-like cells (ICC-L) by conventional transmission electron microscopy (TEM) and immuno-transmission electron microscopy (I-TEM) with antibodies directed towards CD117 and CD34. Two main types of interstitial cells were identified by TEM: ICC-L and fibroblast-like cells (FLC). ICC-L were bipolar with slender (0.04 μm) flattened dendritic-like processes, frequently forming a branching labyrinth network. Caveolae and short membrane-associated dense bands were present. Mitochondria, rough endoplasmic reticulum and Golgi apparatus were observed in the cell somata and cytoplasmic processes. Intermediate filaments were abundant but no thick filaments were found. ICC-L were interconnected by close appositions, gap junctions and peg-and-socket junctions (PSJ) but no specialised contacts to smooth muscle or nerves were apparent. FLC were characterised by abundant rough endoplasmic reticulum but no caveolae or membrane-associated dense bands were observed; gap junctions and PSJ were absent and intermediate filaments were rare. By I-TEM, CD34 gold immunolabelling was present in long cytoplasmic processes corresponding to ICC-L between muscle fascicles but CD117 gold immunolabelling was negative. Thus, ICC-like cells are present in the human detrusor. They are CD34-immunoreactive and have a myoid ultrastructure clearly distinguishable from fibroblast-like cells. ICC-L may be analogous to interstitial cells of Cajal in the gut.     

Two patterns of development of interstitial cells of Cajal in the human duodenum

At the end of the embryonic period of human development, c-kit immunoreactive (c-kit IR) cells identifiable as interstitial cells of Cajal (ICC) are present in the oesophagus and stomach wall. In the small and large bowel, c-kit-IR cells appear later (in the small bowel at 9 weeks, and in the colon at 10-12 weeks), also in the MP region. The object of this study was to determine the timing of appearance and distribution of c-kit IR cells in the human embryonic and foetal duodenum. I used immunohistochemistry to examine the embryonic and foetal duodenum for cells expressing CD117 (Kit), expressed by mature ICC and ICC progenitor cells and CD34 to identify presumed ICC progenitors. Enteric plexuses were examined by way of antineuron-specific enolase and the differentiation of smooth muscle cells was studied using antidesmin antibodies. At the end of the embryonic period of development, c-kit IR cells were solely present in the proximal duodenum in the form of a wide belt of densely packed cells around the inception of the myenteric plexus (MP) ganglia. In the distal duodenum, c-kit IR cells emerged at the beginning of the foetal period in the form of thin rows of pleomorphic cells at the level of the MP. From the beginning of the fourth month, the differences in the distribution of ICC in the different portions of the duodenum were established, and this relationship was still present in later developmental stages. In fact, in the proximal duodenum, ICC of the MP (ICC-MP), ICC of the circular muscle (ICC-CM) and ICC of the septa (ICC-SEP) were present, and in the distal duodenum ICC-MP and ICC-SEP only. In conclusion, in the humans there is a difference in the timing and patterns of development of ICC in the proximal duodenum compared to the distal duodenum.     

Effects of lipopolysaccharide-induced inflammation on the interstitial cells of Cajal

Interstitial cells of Cajal (ICC) have recently been found to display phenotypic changes. The present study is designed to determine whether phenotypic changes occur in ICC associated with an inflammatory microenvironment and whether the ICC phenotype could be recovered after the discontinuation of inflammatory stimuli. Immunohistochemistry studies revealed that the functional ICC marker, c-kit, was markedly reduced in patients with Hirschsprung’s disease (n?=?34) compared with controls (n?=?12), whereas another marker of ICC, CD34, was not altered significantly. Compared with the vehicle group (n?=?6), intraperitoneal injection of lipopolysaccharide (LPS; 1.5 mg/kg) in mice (n?=?6) significantly induced plasma tumor necrosis factor-alpha (TNF-α) levels as determined by enzyme-linked immunosorbent assay. Western blot and real-time polymerase chain reaction assessment further showed that LPS injection markedly suppressed intestinal c-kit protein and mRNA expression, which could be blocked by Toll-like receptor 4 (TLR4) deficiency (n?=?6) rather than TLR2 deficiency (n?=?6) and had no effects on CD34. Compared with the vehicle group (n?=?6), intraperitoneal TNF-α (30 μg/kg) administration (n?=?6) also significantly reduced intestinal c-kit protein and mRNA levels but not CD34 levels. However, the reduction of c-kit induced by TNF-α injection was not suppressed by TLR4 deficiency (n?=?6). Intestinal c-kit protein and mRNA levels were markedly restored after the discontinuation of TNF-α administration for 7 days. Moreover, immunofluorescence analysis of primary ICC further confirmed that exposure to TNF-α for 24 h suppressed c-kit expression, which could be restored after discontinuation of TNF-α exposure. CD34 expression was not altered upon exposure to TNF-α. Thus, phenotypic changes in ICC occur in an inflammatory microenvironment in the gut and LPS, TLR4 and TNFα are crucial to this process.     

Development of dendritic cells in culture from human and murine thymic precursor cells.

The earliest T-precursor population in the adult murine thymus can give rise to dendritic cells (DC) in culture if stimulated with a cocktail of cytokines that includes interleukin (IL)-3, but not with cytokine mixes based on granulocyte-macrophage colony stimulating factor (GM-CSF), normally used to generate myeloid-derived DC. This and other evidence led to the proposal that two different lineages of DC exist, one lymphoid-related and the other myeloid-related. To determine whether this selective response to cytokines was restricted to murine DC, early human thymic T-precursors were isolated and their capacity to generate DC in response to various cytokines directly compared to their murine counterparts. In contrast to cultures of murine thymic precursors, CD34+CD1a- lineage marker negative (Lin-) precursor cells from the human thymus proliferated and generated DC with both the IL-3-containing cytokine mix lacking GM-CSF and with GM-CSF based cytokine mixes. These CD34+CD1a-Lin- human precursor cells also gave rise to NK cells under appropriate culture conditions, but produced no granulocyte, monocyte, eosinophil, megakaryocyte or erythroid cells in standard soft-agar colony-forming cell assays. Thus, although apparently lymphoid-restricted, the human thymic DC precursors responded to the myeloid factor GM-CSF as well as to the cytokines selective for murine lymphoid-related DC.     

c-kit immunocytochemical staining in the cytologic diagnosis of metastatic gastrointestinal stromal tumor. A report of two cases

BACKGROUND: Gastrointestinal stromal tumor (GIST) is a distinct group of mesenchymal neoplasms recently shown to exhibit differentiation toward interstitial cells of Cajal (ICC). C-kit (CD117), an immunocytochemical marker consistently expressed in normal ICC, is demonstrable in 81-100% of GISTs. We report two cases wherein immunocytochemical staining for c-kit aided in the diagnosis of metastatic GIST in the liver. CASES: Two patients, a 37-year-old female (case 1) and a 76-year-old male (case 2), presented with multiple nodules in the liver. They had a history of small bowel GIST resected 11 and 1 year earlier, respectively. Fine needle aspiration of the liver nodules showed loose aggregates or fascicles of spindle cells with elongated to oval nuclei, rare paranuclear vacuoles and eosinophilic cytoplasm. The spindle cells showed minimal (case 1) to moderate nuclear pleomorphism (case 2), with occasional mitotic figures seen in case 2. Immunocytochemical staining revealed strong and diffuse staining for c-kit; it was negative for actin, desmin, CD34 and S-100 protein. Thus, a diagnosis of metastatic GIST was rendered. Histologic review of the primary small bowel GISTs of both cases and the subsequently resected liver nodules in case 1 confirmed the diagnosis. CONCLUSION: Metastatic GIST may pose diagnostic problems due to its broad morphologic spectrum and variable cytologic atypia; in particular, distinction from leiomyosarcoma and other mesenchymal tumors is difficult. The diagnostic difficulty is compounded when the prior history of gastrointestinal tumor is not available or forgotten and when GIST is the initial presentation of the tumor. C-kit is a highly sensitive and reliable immunocytochemical marker that can aid in the diagnosis.