A Case of Granulocyte-Colony Stimulating Factor-Producing Hepatocellular Carcinoma Confirmed by Immunohistochemistry

Granulocyte-colony stimulating factor (G-CSF) is a naturally occurring glycoprotein that stimulates the proliferation and maturation of precursor cells in the bone marrow into fully differentiated neutrophils. Several reports of G-CSF-producing malignant tumors have been published, but scarcely any in the hepatobiliary system, such as in hepatocellular carcinoma (HCC). Here, we encountered a 69-yr-old man with a hepatic tumor who had received right hepatic resection. He showed leukocytosis of 25,450/µL along with elevated serum G-CSF. Histological examination of surgical samples demonstrated immunohistochemical staining for G-CSF, but not for G-CSF receptor. The patient survived without recurrence for four years, but ultimately passed away with multiple bone metastases. In light of the above, clinicians may consider G-CSF-producing HCC when encountering patients with leukocytosis and a hepatic tumor. More cases are needed to clarify the clinical picture of G-CSF-producing HCC

Granulocyte-Colony Stimulating Factor-Producing Pancreatic Adenosquamous Carcinoma Showing Aggressive Clinical Course

Herein, we encountered an 89-year-old woman with pancreatic cancer who presented with fever without infective focus, leukocytosis of 45,860/mu L, and elevation of serum granulocyte-colony stimulating factor (G-CSF). The patient could not receive any curative therapy due to an extremely aggressive clinical course. Specimens taken at necropsy revealed an adenosquamous carcinoma positive for G-CSF by immunohistochemistry; it was only the second reported case to date. She was finally diagnosed with G-CSF-producing pancreatic cancer. In light of the above, clinicians should consider the presence of G-CSF-producing tumors, including pancreatic cancer, when presented with patients showing leukocytosis of unknown origin and fever without infective focus.ArticleINTERNAL MEDICINE. 48(9):687-691 (2009)journal articl

Activation of Six1 Expression in Vertebrate Sensory Neurons.

SIX1 homeodomain protein is one of the essential key regulators of sensory organ development. Six1-deficient mice lack the olfactory epithelium, vomeronasal organs, cochlea, vestibule and vestibuloacoustic ganglion, and also show poor neural differentiation in the distal part of the cranial ganglia. Simultaneous loss of both Six1 and Six4 leads to additional abnormalities such as small trigeminal ganglion and abnormal dorsal root ganglia (DRG). The aim of this study was to understand the molecular mechanism that controls Six1 expression in sensory organs, particularly in the trigeminal ganglion and DRG. To this end, we focused on the sensory ganglia-specific Six1 enhancer (Six1-8) conserved between chick and mouse. In vivo reporter assays using both animals identified an important core region comprising binding consensus sequences for several transcription factors including nuclear hormone receptors, TCF/LEF, SMAD, POU homeodomain and basic-helix-loop-helix proteins. The results provided information on upstream factors and signals potentially relevant to Six1 regulation in sensory neurons. We also report the establishment of a new transgenic mouse line (mSix1-8-NLSCre) that expresses Cre recombinase under the control of mouse Six1-8. Cre-mediated recombination was detected specifically in ISL1/2-positive sensory neurons of Six1-positive cranial sensory ganglia and DRG. The unique features of the mSix1-8-NLSCre line are the absence of Cre-mediated recombination in SOX10-positive glial cells and central nervous system and ability to induce recombination in a subset of neurons derived from the olfactory placode/epithelium. This mouse model can be potentially used to advance research on sensory development

Functional analysis of mSix1-8 in chick.

<p>(A) Schematic diagram of mutation analysis of mSix1-8 enhancer in the DRG. Various mSix1-8-EGFP reporters were co-electroporated into the left side of the neural tube with the control wild-type reporter (mSix1-8wt-mRFP1) at HH14, and fluorescence intensities of EGFP and mRFP1 in the DRG were examined at 48 h.p.e. (B) Results of mutation analysis of mSix1-8 in the DRG. mSix1-8wt-mRFP1 (red) was co-electroporated with various EGFP constructs (green): mSix1-8wt (Ba, Ba'), mSix1-8NR1-4m (Bb, Bb'), mSix1-8TCF/LEFm2 (Bc, Bc'), mSix1-8SMADm (Bd, Bd'), mSix1-8bHLH12m (Be, Be') and mSix1-8POU12m (Bf, Bf'). Numbers in brackets correspond to the bar numbers (1–9) in C. The wild-type reporter (Ba-Bf, Ba') marked the DRG (drg) and weakly the neural tube (nt), while all mutations reduced EGFP levels (Bb'-Bf'). mSix1-8NR1-4m almost completely abolished mSix1-8 enhancer activity (Bb, Bb'). The image is a dorsal view of the trunk region, and anterior is to the left. (C) Quantification of the effect of various mutations on Six1-8 enhancer activity in the DRG. The relative EGFP/mRFP1 levels were calculated for each embryo by measuring five DRG, and are shown relative to the value obtained from the wild-type reporter (100%). Data are mean±SD. The relative EGFP level detected in the DRG that received reporters with various mutations was significantly lower (*<i>p</i><0.001) than that of embryos received wild-type reporter. 1: wild-type (n = 7), 2: NR1m (n = 5), 3: NR2m (n = 6), 4: NR1-4m (n = 8), 5: TCF/LEFm2 (n = 6), 6: SMADm (n = 8), 7: bHLH12m (n = 6), 8: POU12m (n = 6), 9: YYm (n = 7). (D) Schematic diagram of mutation analysis of mSix1-8 enhancer in cranial ganglia. Three mSix1-8-EGFP reporters were co-electroporated into the entire epiblast with the control wild-type reporter (mSix1-8wt-mRFP1) at HH4-5, and the fluorescence intensities of EGFP and mRFP1 in the head region were examined at 48 h.p.e. (E) Mutation analysis of mSix1-8 in cranial ganglia. mSix1-8wt-mRFP1 (red) was co-electroporated with various EGFP constructs (green): mSix1-8wt (Ea, Ea', Ea''), mSix1-8NR1-4m (Eb, Eb', Eb'') and mSix1-8TCF/LEFm2 (Ec, Ec', Ec''). Numbers in brackets correspond to the bar numbers in F. The wild-type reporter (Ea-Ec, Ea') marked the trigeminal ganglion (V), geniculate (VII)/vestibuloacoustic (VIII) ganglion complex (VII/VIII) and weakly the otic vesicle (ov), while the two mutations reduced EGFP levels (Eb'-Ec'). mSix1-8NR1-4m almost completely abolished mSix1-8 enhancer activity (Eb, Eb'). mSix1-8wt shows weak enhancer activity in the posterior optic cup (opc, Eb and Ec). Each image is a lateral view of the left side of the head/neck. Anterior is to the left and dorsal is to the top. (F) Quantification of the effect of the two mutations on mSix1-8 enhancer activity in the trigeminal ganglion. The relative EGFP/mRFP1 levels were calculated for each embryo by measuring trigeminal ganglia of both sides, and are shown relative to the value obtained from the wild-type reporter (100%). Data are mean±SD. The relative EGFP level detected in trigeminal ganglia that received reporters with the two mutations was significantly lower (*<i>p</i><0.001) than that of embryos received wild-type reporter. 1: wild-type (n = 5), 2: NR1-4m (n = 8), 3: TCF/LEFm2 (n = 7). drg: dorsal root ganglia, nt: neural tube, opc: optic cup, ov: otic vesicle, V: trigeminal ganglion, VII/VIII: VII/VIII ganglion complex. Scale bars: 0.5 mm.</p

Pattern and specificity of mSix1-8-NLSCre-mediated recombination in embryos.

<p>(A) The structure of a transgene used to generate the mSix1-8-NLSCre transgenic mouse line. mSix1-8 (538 bp) is placed upstream of the tkintron (the HSV thymidine kinase gene promoter and chimeric intron) to drive the expression of NLSCre. The polyA signal is from SV40. The entire expression unit is flanked by two tandem copies of the core region of the HS4 insulator (ins). The positions of the genotyping PCR primers (arrowheads) and the size of the PCR product (406 bp) are shown. Selected restriction sites are also indicated. (B-H) Localization of ß-Gal-positive cells in mSix1-8-NLSCre/R26R-LacZ double transgenic embryos. At E9.0 (Ba), the earliest sign of the appearance of ß-Gal-positive cells was detected in the otic pit region (white square bracket). A close-up view (Bb) shows signals in three scattered cells (black arrowheads) in the otic pit. A section through the posterior part of the otic pit confirmed the presence of ß-Gal-positive cells in the pits of both sides (Bc). At E9.5 (Ca), ß-Gal-positive cells were noted in the developing trigeminal ganglion (black square bracket) and olfactory placode (yellow square bracket) in addition to the otic pit (white square bracket). A close-up view (Cb) shows signals in scattered cells in and around the olfactory placode (white arrowheads). At E9.75 (Da), signals were detected in the developing geniculate ganglion. In a close-up view (Db) showed signals in the ventral portion of the otic vesicle (black arrow). At E10.5 (Ea), clear signals were detected in all the cranial sensory ganglia (V, VII, VIII, IX and X), cells in and around the olfactory epithelium (Eb, a close-up view) and in the DRG. At E11.0 (F), the intensity of the signals in the sensory organs became stronger. Signals were also found in the mesenchyme of forelimb bud (Ea, F-H), hindlimb bud (F-H), branchial arches (Ea, Eb, F-H), and the maxillary process (F-H). In all panels of whole mount embryos, anterior is to the left, dorsal is to the top, and all panels are lateral views. In the transverse section shown in Bc, dorsal is to the top. ba: branchial arches, drg: dorsal root ganglia, fb: forebrain, fl: forelimb bud, hb: hindbrain, hl: hindlimb bud, mp: maxillary process, oe: olfactory epithelium, op: olfactory placode, otp: otic pit, ov: otic vesicle, V: trigeminal ganglia, VII: geniculate ganglia, VII/VIII: VII/VIII ganglion complex, IX: petrosal ganglion, X: nodose ganglion. Scale bars: 2 mm (H), 1 mm (Ba, Ca, Da, Ea, F, G), 0.2 mm (Bb), 0.5 mm (Cb, Db, Eb), 0.1 mm (Bc).</p

Functional analysis of mSix1-8 in mouse.

<p>(A) Mutation analysis of mSix1-8 in mouse. Wild-type (mSix1-8wt-LacZ, Aa) and mutated [(mSix1-8NR1-4m-LacZ, Ab) and (mSix1-8TCF/LEFm2-LacZ, Ac)] transgenes were used for transgenesis and ß-Gal localization was examined at E10.5. Embryos injected with the wild-type transgene showed ß-Gal activity specifically in the trigeminal ganglion, the VII/VIII ganglion complex and epibranchial placode/ganglia (Aa)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136666#pone.0136666.ref017" target="_blank">17</a>]. However, ß-Gal activity was almost completely lost in an embryo carrying NR1-4m mutation, with the exception of a small number of ectopic ß-Gal-positive cells in the cervical area (Ab). In an embryo carrying TCF/LEFm2 mutation, ß-Gal activity was lost in the sensory organs and ganglia, with the exception of ectopic activity in the somites (Ac). drg: dorsal root ganglia, so: somites, V: trigeminal ganglion, VII/VIII: VII/VIII ganglion complex, IX: petrosal ganglion, X: nodose ganglion. Scale bars: 1 mm. (B) Summary of the phenotypes of transgenic mouse embryos. transgenic embryos: the total number of transgenic embryos obtained using each transgene, pattern A: number of embryos with ß-Gal staining pattern in which the signal in the trigeminal ganglion stands out as the major ß-Gal-positive domain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136666#pone.0136666.g003" target="_blank">Fig 3Aa</a> represents a typical pattern A staining), pattern B: number of embryos with ß-Gal staining in which signals in the cranial ganglia was reduced while those in the DRG were relatively unaffected, embryos with ectopic expression: number of embryos with an ectopic LacZ staining, embryos without sensory ganglia-expression: number of embryos without a LacZ staining in the sensory ganglia. Parts of the results obtained using the wild-type transgene were reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136666#pone.0136666.ref017" target="_blank">17</a>].</p