Clinicopathologic and Surgical Characteristics Study of 151 Cases of GIST.

BACKGROUND: Gastrointestinal stromal tumor (GIST) is the most appearing mesenchymal tumor of the gastrointestinal system. In this study, we are aiming to share the most up to date knowledge about diagnosis and treatment of these tumors by transferring our clinical experience about GISTs. METHODS: The 151 patients who were operated between 2006-2020 and whose pathological examination was reported as GIST were analyzed retrospectively. Demographic, clinical, and pathological features and treatment methods of patients were evaluated. RESULTS: Seeventy-six of the patients were women and 75 of them were men whose age averages were 66.1 (31-86). The most common location was the stomach (55.6%), followed by the small intestine, retroperitoneal, large intestine, rectum, esophagus, and another organ. With surgical intervention, 139 of them had been cured. Twelve of cases were accepted as inoperable. The diameter of tumors in our cases were between 0.4 cm and 35 cm. Determined mitotic activity was ≤ 5 in 71 patients and 5 < in 80 patients. In 8 of 12 unresectable cases, it has been seen that partial remission after the treatment of 12-month tyrosine kinase inhibitors, C-KIT, was positive in 96.7% of our cases. CD34 and Ki-67 was analyzed in patients. CD34 was found positive in 98 (64.9%) of them, Ki-67 was positive in 82 (54.3%) patients. Patients had been observed for 40 months. CONCLUSION: Despite GISTs are not appearing frequently, nowadays they have started to be seen more frequently than before with the growing present-day diagnostic methods. The ideal treatment is performing radical resection without leaving any tumor cells behind. Tyrosine kinase inhibitors have an important place in unresectable cases.

Mouse intestinal niche cells express a distinct α1,2-fucosylated glycan recognized by a lectin from Burkholderia cenocepacia.

In this study, we examined the distribution of fucosylated glycans in mouse intestines using a lectin, BC2LCN (N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia), as a probe. BC2LCN is specific for glycans with a terminal Fucα1,2Galß1,3-motif and it is a useful marker for discriminating the undifferentiated status of human induced/embryonic stem cells. Apparent BC2LCN reactivity was detected in the secretory granules of goblet cells in the ileum but not those in the colon. We also found distinctive reactivity in the crypt bottom, which is known as the stem cell zone, of the colon and the ileum. Other lectins for fucosylated glycans, including Ulex europaeus agglutinin-I, Pholiota squarrosa lectin and Aleuria aurantia lectin, did not exhibit similar reactivity in the crypt bottom. Remarkably, BC2LCN-positive epithelial cells could be labeled with a niche cell marker, c-Kit/CD117. Overall, our results indicate that intestinal niche cells express distinct fucosylated glycans recognized by BC2LCN. Increasing evidence suggests that the self-renewal and proliferation of stem cells depend on specific signals derived from niche cells. Our results highlight novel molecular properties of intestinal niche cells in terms of their glycosylation, which may help to understand the regulation of intestinal stem cells. The distinct expression of glycans may reflect the functional roles of niche cells. BC2LCN is a valuable tool for investigating the functional significance of protein glycosylation in stem cell regulation.

c-kit mutational analysis in paraffin material.

The current WHO classification of mastocytosis defines one major and four minor diagnostic criteria for systemic mastocytosis (SM). One of the minor criteria is the detection of the "gain-of-function" mutation D816V of the c-kit proto-oncogene in extracutaneous organs. The receptor molecule KIT is a potential therapeutic target for tyrosine kinase inhibitors. KIT mutations have been described in more than 80% of SM, but only in the minority of cutaneous mastocytoses (CM). Usually exon 17 amplicons generated by polymerase chain reaction are analyzed for the detection of c-kit mutations. Most frequently the method of restriction fragment length polymorphism (RFLP) analysis using the endonuclease Hinf I is used. Another well-established technique utilizes melting point analysis of amplification products with specific hybridization probes. Recently, also allele-specific PCR assays have been described. The technique used for the detection of c-kit mutations in mastocytosis is dependent on the kind of material to be analyzed and the laboratory equipment available. In this chapter the techniques of PNA-mediated PCR-clamping in combination with melting point analysis for the genotyping of amplification products are described for mutational analysis in total DNA and microdissected cells from formalin-fixed paraffin-embedded bone marrow trephine biopsies.

Extragonadal seminoma presenting as a large mass in the pelvic cavity without c-kit-activating mutations.

Extragonadal germ cell tumors are relatively rare tumors, which usually occur in the mediastinum or retroperitoneum. In this report, we present a case of primary seminoma arising in the pelvic cavity. A 58-year-old man with urinary retention and abdominal distension was admitted to our hospital. Computed tomography and magnetic resonance imaging demonstrated a large mass in the pelvic cavity. Histological examination of the specimens obtained by open biopsy revealed seminomatous malignant cells. Immunohistochemical studies detected vimentin, placental alkaline phosphatase and c-kit. Taking these results together with the patient's other clinical manifestations, this case was diagnosed as extragonadal seminoma without c-kit-activating mutations, and chemotherapy followed by radiation therapy was successful. Primary seminoma in the pelvic cavity is extremely rare, but should be considered a cause of pelvic mass formation.

Avances en los tumores del estroma gastrointestinal / A review of the recent advances in gastrointestinal stromal tumours

Los tumores del estroma gastrointestinal (GIST) son las neoplasias mesenquimales más comunes del tubo digestivo y representan uno de los mejores modelos de tratamiento farmacológico dirigido a dianas moleculares específicas. En general, el diagnóstico morfológico no plantea mayores problemas debido a que el cuadro histológico (celularidad fusiformes y/o epiteliode) e inmunohistoquímico (expresión de CD117, CD34 y, más eventualmente, de actina de músculo liso, desmina e incluso proteína S-100) suele ser bastante característico. No obstante, en el 5–10% de los casos la histología y los resultados inmunohistoquímicos no son los esperados y el diagnóstico debe descansar en la demostración mediante técnicas de biología molecular de mutaciones en KIT o PDGFRA, ya que el diagnóstico correcto es requisito necesario para la aplicación de la terapia específica. En el presente artículo llevamos a cabo una revisión sobre los avances acontecidos en 5 aspectos fundamentales sobre la biología y diagnóstico de estas neoplasias: nuevos marcadores inmunohistoquímico, factores pronósticos, biología molecular, síndromes clínicos asociados y respuesta tisular a los inhibidores de tirosin quinasa(AU)

Gastrointestinal stromal tumours (GIST) are the most common mesenchymal neoplasms of the intestinal tract and are one of the best models for treatment with molecular target therapy. Morphological diagnosis does not usually present many problems due to the presence fusiform and/or epitheliod cells together with the characteristic immunohistochemical expression of CD117, CD34 and, less frequently, smooth muscle actin, desmin and even S-100 protein. However, unexpected histological and immunohistochemical results are found in 5 to 10% of cases. In such unusual cases, molecular biology is needed to demonstrate c-KIT or PDGFRA mutations in order to make a correct diagnosis, which is a necessary prerequisite for molecular target therapy. The present article reviews recent advances in five fundamental biological and diagnostic aspects of GIST: new immunohistochemical markers, prognostic factors, molecular biology, associated clinical syndromes and tissue response to tyrosine kinase inhibitors(AU)

Histological and functional organization in human testicle: expression of receptors c-kit and androgens / Organización histológica y funcional en el testículo humano: expresión de los receptores c-kit y andrógenos

The objective of this work was identify the presence of interstitial cells of Cajal, muscle cells, nerves and androgen receptor positive cells in adult human testicle, using immunohistochemical detection for c-kit/CD-117, actin smooth muscle specific (ASMS), neurofilament (N) and androgen receptor (AR), respectively. The samples were obtained from patients (n= 10) with diagnosis of prostate cancer, with surgery of orchiectomy. Subsequently were processed by histology and for immunohistochemistry using specific antibodies. It showed the presence of cells c-kit/CD-117, with diverse degrees of positivity, distributed mainly in the interstitial peritubular area of the human testicle. The peritubular myoides cells were positive to the presence of the actin smooth muscle and androgen receptor. The neurofilaments elements (+) only were observed in the vascular tunic. The specific immunohistochemistry describe the presence of the interstitial cells of Cajal in human testicular interstitium, opening a new perspective for the functional interpretation of the testicular cellularity and tubular motility. Possibly associated functionally to peribubulars cells of smooth muscle to regulate the mobility of the seminiferous tabules, whose integration and function would be androgen dependent. The cells that express the c-kit receptor, were found exclusively in the interstitial compartment. This cellular type in addition of the muscular cells of peritubules and the absence of nervous fibers to the interior of the testicle, could be responsible for the regulation of tubular mobility, as it happens in the gastrointestinal apparatus.

El objetivo de este trabajo fue identificar la presencia de células interticiales de Cajal, células musculares lisas, células nerviosas y células que expresan receptores de andrógeno en el testículo de humano adulto, usando inmunohistoquímica específica para: c-kit/CD-117, músculo liso actina específico (ASMS), neurofilamentos (N) y para receptores de andrógenos (AR). Las muestras fueron obtenidas de pacientes (n=10) con diagnóstico de cáncer prostático sometidos a cirugía de orquiectomía. Las biopsias se procesaron para histología e inmunohistoquímica usando anticuerpos específicos. Se muestra la presencia de células c-kit/CD-117, con diversos grados de positividad y distribuidas en el compartimento interticial del testículo. Las células peritubulares mioides fueron positivas para la presencia de músculo liso actina específico y para receptor de andrógenos. La marcación de neurofilamentos positivos, sólo fueron observados en la túnica vascular. Conclusiones: La inmunohistoquímica específica describe la presencia de células interticiales de Cajal en los interticios testiculares humanos, abriendo una nueva visión en la interpretación funcional de la celularidad testicular y la motilidad tubular. Lo anterior asociado a la funcionalidad de las células peritubulares (músculo liso) regularían la motilidad de los túbulos seminíferos. Este proceso posiblemente es andrógeno dependiente. Las células que expresan receptores c-kit se encuentran exclusivamente en los compartimentos interticiales, estas células en conjunto con las células musculares peritubulares agregado a la ausencia de fibras nerviosas al interior del testículo, podrían ser los responsables de la regulación de la motilidad tubular, similar a como se informa para el tracto gastrointestinal.

Tumores del estroma gastrointestinal (GIST): factores pronósticos de supervivencia tras citorreducción R0 / No disponible

Objetivo: analizar los posibles factores pronósticos de supervivenciaen tumores estromales gastrointestinales c-kit positivo(GIST), tras citorreducción óptima R0.Pacientes y método: estudio de 35 pacientes intervenidosen nuestra Unidad desde enero 2002 a febrero 2007, con tumoresdel estroma gastrointestinal CD117/c-kit positivo en los quese alcanzó citorreducción quirúrgica sin residuo tumoral macroscópico.Una base de datos prospectiva nos proporcionó las distintasvariables analizadas, de carácter demográfico, anatómico, clínico,histopatológico e inmunohistoquímico, entre otras. Elanálisis de la supervivencia actuarial se realizó según el método deKaplan-Meier y el análisis multivariante mediante el método de regresiónmúltiple de Cox.Resultados: la supervivencia global a 5 años fue del 77%,con una supervivencia media de 52 meses. El riesgo de malignidadsegún la clasificación de Fletcher y el tamaño tumoral mayorde 10 cm, influyeron significativamente de forma negativa sobrela supervivencia de los pacientes, tras el análisis univariante realizado(p < 0,05). La actividad proliferativa Ki-67 mayor del 50%fue la única covariable con significación estadística en el análisismultivariante. El 20% de los tumores recurrieron. Sólo 3 pacientesmetastáticos recibieron tratamiento adyuvante con mesilato deimatinib, todos ellos con Ki-67 > 50% y vivos en la actualidad.Conclusiones: el índice proliferativo Ki-67 podría representarun excelente marcador pronóstico de supervivencia en aquellospacientes con tumores del estroma gastrointestinal c-kit positivo.Su confirmación y el punto de corte adecuado deberían serobjeto de futuros estudios prospectivos, así como su posible utilidadpara seleccionar pacientes candidatos al tratamiento con mesilatode imatinib

Objective: to analyze the different factors predictive of survivalassociated with optimal R0-cytoreduction in c-kit-positivegastrointestinal stromal tumors.Methods: thirty-five patients were operated on in our OncologicalSurgery Department from January 2002 to February2007 because of CD117/c-kit-positive gastrointestinal stromal tumors,and an optimal surgical cytoreduction was obtained withoutmacroscopical residual disease. Demographic, anatomical, clinical,pathological, and immunohistochemical variables were analyzedfrom a specific database. Survival and multivariate analyseswere developed using Kaplan-Meier and multiple Cox regressionmodels, respectively.Results: five-year overall survival was 77% with a mean survivalof 52 months. Risk of malignant behaviour according toFletcher’s classification and tumor size higher than 10 cm had asignificantly negative influence on overall survival in the univariateanalysis (p < 0.05). Proliferative Ki-67 activity higher than 50%was the only statistically significant variable in the multivariateanalysis. Twenty percent of tumors recurred. Only 3 patients withmetastatic disease received adjuvant treatment with imatinib mesylate,all of them with Ki-67 > 50% and currently alive.Conclusions: the poliferative Ki-67 index could represent anexcellent predictive factor for survival in patients with c-kit-positivestromal gastrointestinal tumors. Confirmation and an adequatecut-off level should be the main objectives for futureprospective studies, mostly focused on the appropriate selectionof optimal candidates to imatinib-mesylate-based treatment

[Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor].

OBJECTIVE: To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF). METHODS: In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay. RESULTS: In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L. CONCLUSION: c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.

ChT1, an Ig superfamily molecule required for T cell differentiation.

The thymus is colonized by circulating progenitor cells that differentiate into mature T cells under the influence of the thymic microenvironment. We report here the cloning and function of the avian thymocyte Ag ChT1, a member of the Ig superfamily with one V-like and one C2-like domain. ChT1-positive embryonic bone marrow cells coexpressing c-kit give rise to mature T cells upon intrathymic cell transfer. ChT1-specific Ab inhibits T cell differentiation in embryonic thymic organ cultures and in thymocyte precursor cocultures on stromal cells. Thus, we provide clear evidence that ChT1 is a novel Ag on early T cell progenitors that plays an important role in the early stages of T cell development.

Characterization of prethymic progenitors within the chicken embryo.

The thymic primordium in both birds and mammals is first colonized by cells emerging from the intra-embryonic mesenchyme but the nature of these precursors is poorly understood. We demonstrate here an early embryonic day 7 prethymic population with T lymphoid potential. Our work is a phenotypic analysis of, to date, the earliest embryonic prethymic progenitors arising in the avian para-aortic area during ontogeny. The phenotype of these cells, expressing the cell surface molecules alpha2beta1 integrin, c-kit, thrombomucin/MEP21, HEMCAM and chL12, reflects functional properties required for cell adhesion, migration and growth factor responsiveness. Importantly, the presence of these antigens was found to correlate with the recolonization of the recipient thymus following intrathymic cell transfers. These intra-embryonic cells were also found to express the Ikaros transcription factor, the molecular function of which is considered to be prerequisite for embryonic lymphoid development.

A transforming mutation enhances the activity of the c-Kit soluble tyrosine kinase domain.

An activating mutation (DY814) located in the catalytic domain of the c-Kit receptor has been found in mastocytomas from human, mouse and rat. We evaluated the enzymic properties of purified wild-type (WT) and DY814 tyrosine kinase domains expressed in Pichia pastoris. A linker encoding the Flag epitope was fused to c-Kit cDNA species, enabling affinity purification of the proteins with anti-Flag antibodies. Yeast lysates expressing DY814 contained multiple tyrosine-phosphorylated proteins, whereas WT lysates had no detectable tyrosine phosphorylation. Purification of the WT and mutant kinases in the presence of vanadate demonstrated that both enzymes undergo autophosphorylation. Kinetic analyses of WT and DY814 kinases indicated that at 20 nM enzyme concentration the mutation increases the specific activity 10-fold and decreases the apparent Km for ATP 9-fold. WT activity displayed a hyperbolic dependence on enzyme concentration, consistent with a requirement for dimerization or aggregation for activity. This activity was also enhanced by anti-Flag antibodies. In contrast, the dependence of DY814 activity on enzyme concentration was primarily linear and only marginally enhanced by anti-Flag antibodies. Gel-filtration analysis showed that the WT kinase migrated as a monomer, whereas the DY814 mutant migrated as a dimer. These results indicate that this point mutation promotes dimerization of the c-Kit kinase, potentially contributing to its transforming potential in mast cells.

Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis.

The Kit receptor and its ligand KL, which together constitute an essential effector at various stages of embryonic development, are both present during adult gametogenesis. In the testis, KL is expressed in Sertoli cells, and Kit in germ cells, starting at the premeiotic stages. A series of observations indicated previously a role in spermatogonia survival, without excluding a possible function at later stages. We identified a complex pattern of expression of the two components in the adult murine testis, suggestive of a role in the meiotic progression of spermatocytes. At stages VII-VIII of the cycle of the seminiferous epithelium, the time when spermatocytes enter meiosis, the membrane-associated form of KL extends on the Sertoli cell from the peripheral to the adluminal compartment of the tubule. We also found that the receptor is present on the surface of germ cells up to the pachytene stage. The availability of differentiated Sertoli cell lines, which express the KL protein and support part of the maturation of germ cells in coculture, allowed us to ask whether, in the in vitro reconstructed system, transit of spermatocytes through meiosis requires the Kit-KL interaction. Addition of a blocking monoclonal antibody against the Kit receptor (ACK2) inhibited extensively the appearance of haploid cells and the expression of a haploid-phase-specific gene (Prm1). Recognition of the supporting Sertoli cell by germ cells was not affected, indicating a requirement for the activity of the receptor for either entering or completing meiosis. Involvement of the membrane-associated form of the ligand was suggested by the observation that addition of the soluble form of KL was equally inhibitory.

Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells.

Stem cell factor (SCF) is a cytokine critical for normal hematopoiesis. The receptor for SCF is c-Kit, a receptor tyrosine kinase. Our laboratory is interested in delineating critical components of the SCF signal transduction pathway in hematopoietic tissue. The present study examines activation of Src family members in response to SCF. Stimulation of cell lines as well as normal progenitor cells with SCF rapidly increased tyrosine phosphorylation of the Src family member Lyn. Peak responses were noted 10-20 min after SCF treatment, and phosphorylation of Lyn returned to basal levels 60-90 min after stimulation. SCF also induced increases in Lyn kinase activity in vitro. Lyn coimmunoprecipitated with c-Kit, and studies with GST fusion proteins demonstrated that Lyn readily associated with the juxtamembrane region of c-Kit. Treatment of cells with either Lyn antisense oligonucleotides or PP1, a Src family inhibitor, resulted in dramatic inhibition of SCF-induced proliferation. These data demonstrate that SCF rapidly activates Lyn and suggest that Lyn is critical in SCF-induced proliferation in hematopoietic cells.

Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo.

At day 10 in mouse gestation, the intraembryonic aorta-gonads-mesonephros (AGM) region generates the first definitive hematopoietic stem cells (HSCs) of the adult blood system. By 11 days postcoitum, the liver contains such HSCs. While HSCs of the adult bone marrow and late-stage fetal liver have been extensively characterized for cell surface markers, there has been no phenotypic description of the first HSCs during embryo development. We report here the temporal cell surface phenotype of HSCs from the AGM region and early fetal liver and show that all HSCs reside in the c-kit+ population. c-kit+ HSCs from AGM and liver are mainly CD34+ and in the AGM are in both Mac-1+ and Mac-1 fractions. These results demonstrate that during mouse ontogeny the first definitive HSCs are similar in cell surface phenotype to the HSCs of adult bone marrow but that spatial localization and developmental time are critical factors in the phenotypic assessment of this functional cell population.

Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop.

We have revealed that about one and a half thousand tiny clusters, filled with one thousand closely packed lymphocytes, can be found throughout the murine small and large intestinal mucosa. They are located in crypt lamina propria (cryptopatches; CP) and can be first detected at 14-17 d after birth. A large fraction of lymphocytes in CP expresses c-kit, IL-7R, Thy1 and a lymphocyte function-associated antigen, LFA-1, whereas most of them remain CD3-, TCR alpha beta-, TCR gamma delta-, sIgM-, and B220-. The population size of IL-2R alpha+, HSA+ and Pgp-1+ subsets is variable (20-50%) and the composition of CD8+, Ly-1+, and CD4+ subsets is smaller but also variable (3-20%). In the small intestine, CP do not contain cells undergoing apoptosis nor cells bearing RAG-1 molecules, but do contain dendritic stromal cells bearing CD11c/CD18 molecules. The frequency of DNA replicating cells in CP is higher than that in Peyer's patches (PP), is lower than that in the thymic cortex and is almost comparable with that in the thymic medulla. The numbers of CP remain the same in aged mice (> 114 wk) but double after estrogen treatment even though the thymi are attenuated sharply in both conditions. Thus, with respect to histogenesis, lymphocyte composition and tissue level of cellular behavior, neither PP, isolated lymphoid follicles, peripheral LNs, nor thymus are identical with CP. Finally, CP are virtually absent in lamina propria of IL-7R-deficient mice that display a profound reduction in thymic and peripheral lymphoid cellularity. By contrast, CP are present in germ-free mice and in athymic (nu/nu), SCID, TCR beta x delta-/-, RAG-2-/-, PP-deficient (aly/aly), stem cell factor (Sl/Sld) and c-kit (W/Wv) mutant mice. Taking all of these results together, CP are the first identification of gut-associated murine lymphoid tissues where the generation of IL-7-dependent lympho-hematopoietic progenitors for T and/or B cell descendants may start to take place at the age of commencement of weaning.

Expression of multiple c-kit receptor messenger ribonucleic acid transcripts during postnatal development of the rat testis.

The c-kit protooncogene is a transmembrane tyrosine kinase receptor expressed during gametogenesis. Using the polymerase chain reaction (PCR), we have identified the c-kit receptor mRNA transcripts in the rat testis and studied their expression during postnatal development of the testis. Five different transcripts were identified using sets of primers encoding within the extracellular domain. Two transcripts were obtained from primer sets encoding regions within the cytoplasmic domain and the primer set encoding the entire length of the c-kit receptor. We have compared the levels of expression of these transcripts on different days during postnatal development. The level of expression of a particular transcript varied depending upon the developmental stage of the testis. In summary, our results suggest that multiple forms of mRNAs exist for the c-kit receptor in the rat testis, and they are regulated differentially during postnatal development.