Methotrexate-related lymphoproliferative disorder of the stomach in a patient with rheumatoid arthritis: a case of disease regression after methotrexate cessation.

We report the case of a 78-year-old woman with methotrexate-related gastric lymphoproliferative disorder (LPD). The patient had a history of rheumatoid arthritis (RA) and had been treated with methotrexate (MTX). Endoscopic examination revealed round elevated lesions in the stomach, and a biopsy specimen showed atypical lymphoid cell proliferation. Immunohistological study found these atypical cells to be positive for L-26 but not for CD3 or EBER. Therefore, we made a diagnosis of MTX-related LPD showing features of diffuse large B-cell lymphoma. Combined positron emission tomography-computed tomography (PET-CT) using 18F-fluorodeoxyglucose (FDG) showed increased avidity in the stomach in addition to slightly increased FDG-avidity in the mediastinum and left chest wall. We decided not to start chemotherapy but to discontinue administration of MTX, with follow-up using endoscopy and PET-CT. The endoscopic examinations after cessation of MTX demonstrated gradual regression of the elevated lesions. PET-CT 6 months after cessation showed no increased FDG avidity in the stomach. While disease regression was observed in the stomach, the other FDG-avid spots remained unchanged on PET-CT. Therefore, we performed chemotherapy as additional therapy. On PET-CT after chemotherapy, the FDG-avid spots remained unchanged for more than 1 year, and we eventually concluded that they were RA-related inflammatory lesions. In patients with MTX-related LPD, cessation of MTX may be a therapeutic option, but careful follow-up and chemotherapy in accordance with the clinical course are essential.

[Hepatic Resection of Multiple Liver Metastases from Gastric Cancer after Molecular Targeted Chemotherapy(S-1 plus Cisplatin plus Trastuzumab)].

A 62-year-old man was diagnosed with gastric cancer and underwent distal gastrectomy, and D1+b lymph node dissection. He was diagnosed postoperatively with T1b (sm2) N0M0, StageⅠA gastric adenocarcinoma and did not receive any adjuvant chemotherapy after surgery. One year and 6 months after gastrectomy, blood analysis indicated high levels of carcinoembryonic antigen (CEA 262.1 ng/mL) while abdominal computed tomography (CT) revealed multiple liver tumors (S7: 15 mm, S7/8: 20 mm). The patient was diagnosed with metachronous multiple liver metastases from gastric cancer. Chemotherapy, combined with molecular targeted therapy (S-1 plus cisplatin [CDDP] plus trastuzumab), was administered because of overexpression of the human epidermal growth factor receptor 2 (HER2) protein in the primary tumor as assessed by immunohistochemistry, the CEA levels decreased immediately after 2 cycles of the chemotherapy, and the liver metastases shrank markedly with no evidence of new lesions on abdominal CT. However, after treatment, Grade 3 neutropenia and diarrhea were observed. Chemotherapy was suspended and hepatic resection was performed. After hepatic resection, the liver tumors were histologically evaluated as Grade 2 metastatic gastric adenocarcinoma, and the HER2 expression of remnant carcinoma cells was established. The patient has been in good health and remained free of recurrences in the 2 years and 3 months after the liver resection. Surgery with preoperative chemotherapy (S-1 plus CDDP plus trastuzumab) can be an effective treatment for liver metastasis from HER2-positive gastric cancer.

[A Case of HER2-Positive Advanced Gastric Cancer with a Pathological Complete Response to Neoadjuvant Chemotherapy with S-1/CDDP/Trastuzumab].

A 61-year-old man was referred to our hospital because of epigastric pain. Upper gastrointestinal endoscopy revealed a type 2 tumor at the gastric antrum, which was diagnosed as gastric adenocarcinoma (tub1) by pathological examination and was HER2 positive 3+ by the IHC method. Abdominal computed tomography revealed multiple metastases to regional lymph nodes (LNs), including bulky nodes at No. 3, 6, and 11p stations. In particular, No. 6 LN was 43 mm in diameter and had invaded to the pancreas. The clinical stage was Ⅲc (T4aN3M0) and neoadjuvant chemotherapy was conducted using S-1/CDDP/trastuzumab. After 2 cycles of chemotherapy, a partial clinical response was obtained and distal gastrectomy with LN dissection (D2 plus No. 16 LN) was performed. The pathological specimens showed no residual cancer cells in the stomach and LNs (Grade 3: pCR). Adjuvant chemotherapy was not administered. The patient is alive 10 months after the surgery with no evidence of recurrence.

[A Case of Conversion Surgery after Long-Term Chemotherapy for Advanced Gastric Carcinoma with Synchronous Distant Metastasis].

We report the case of a patient who underwent conversion surgery after long-term chemotherapy for advanced gastric carcinoma with synchronous distant metastasis. She was admitted to our hospital because of back pain and elevated serum ALP level. Gastrointestinal endoscopy showed multiple 0-Ⅱa like lesions at the gastric antrum, and a biopsy specimen showed poorly differentiated adenocarcinoma negative for HER2. Colonoscopy showed a submucosal tumor at the cecum, and pathological examination revealed metastatic gastric adenocarcinoma. CT revealed regional lymph node metastasis, bilateral ovarian tumors, and systemic bone absorption indicating metastasis. Systemic chemotherapy with cisplatin and S-1 was carried out, and complete resolution of gastric and colic lesions was obtained. Afterwards, a new gastric lesion appeared with re-growth of regional lymph nodes and bilateral ovarian tumors. Distal gastrectomy with D2 dissection and bilateral ovariectomy was performed 2 years 6 months after the initial therapy. Although systemic chemotherapy is the standard treatment for advanced gastric carcinoma with distant metastasis, surgical resection can be justified when drug-resistant lesions are localized and conversion surgery is feasible. Postoperative chemotherapy is mandatory to prolong survival.

[A Case of Bladder Adenocarcinoma with Rectal Metastasis].

A 77 year-old man with asymptomatic microscopic hematuria underwent a cystoscopic examination, which identified a broad-based papillary tumor at the cervix of the bladder. Adenocarcinoma was detected in the biopsy specimen. MRI and CT examination showed a huge papillary tumor of the bladder invading the inner lobe of the prostate. In addition, the wall of the lower rectum exhibited thickening with enlargement of the regional lymph nodes. Endoscopy disclosed a hemi-circular rectal tumor and pathological examination revealed adenocarcinoma, the profile of which was similar to the bladder tumor. The levels of CEA and CA19-9 were 5.3 ng/mL and 39 U/mL, respectively. A differential diagnosis considering bladder cancer, rectal cancer, or both was necessary before planning a treatment strategy. Since both tumors were judged to be resectable, total pelvic exenteration was carried out. Through detailed postoperative pathological examinations, it was concluded that this tumor was of bladder origin and it invaded the prostate along with metastasis to the rectum. Adenocarcinoma of the bladder is extremely rare and exhibits aggressive behavior.

Medium osmolarity-dependent biosynthesis of renal cellular sulfoglycolipids is mediated by the MAPK signaling pathway.

Verots S3 and Vero317 cells were shown by metabolic labeling with (35)S-sulfate to contain many more sulfoglycosphingolipids than original Vero cells derived from African green monkey kidney. The activity of galactosyl ceramide sulfotransferase (GST) was shown to be 89- and 92-fold higher in Vero317 cells and Verots S3 cells, respectively, than that of the parent cells, whereas the activity of the degradation enzyme, arylsulfatase A, was unchanged among all the three cell strains. GST gene transcript levels in Verots cells were 14.3-fold higher than those in Vero cells. The cell adhesiveness to the culture plate under hypertonic stress was strengthened significantly in both mutant strains. Among the major sulfoglycolipids of the Verots S3 cell line, assigned as SM4s, SM3, SM2a, and SB1a, the incorporation of (35)S-sulfate into SM3, SM2a and SB1a was upregulated with the increasing tonicity of the medium. Sulfoglycolipids in these renal cells seemed to contribute to the membrane barrier against hypertonic media as shown previously in another renal cell line, MDCK (Niimura and Nagai, 2008). Sulfoglycolipid synthesis was suppressed with the p38 (MAPK) inhibitor SB203580 and/or with the MEK-1/2 (MAPKK) inhibitor PD98059, and with the tyrosine kinase inhibitor genistein, which also reduced the sulfoglycolipid synthesis in a dose-dependent manner. Further the administration of the MAPK/MAPKK inhibitors to the culture medium reduced significantly the viability of Verots S3 cells under hypertonic stress. These findings suggest that sulfoglycolipid synthesis in those renal cells may be regulated to adapt to the renal osmotic circumstances by the medium's osmolarity via the MAPK signaling pathway.

Long-term culture following ES-like gene-induced reprogramming elicits an aggressive phenotype in mutated cholangiocellular carcinoma cells.

BACKGROUND: We recently reported that gastrointestinal (GI) cancer cells can be reprogrammed to a pluripotent state by the ectopic expression of defined embryonic stem (ES)-like transcriptional factors. The induced pluripotent cancer (iPC) cells from GI cancer were sensitized to chemotherapeutic agents and differentiation-inducing treatment during a short-term culture, although a phenotype induced by long-term culture needs to be studied. METHODS: A long-term cultured (Lc)-iPC cells were produced in GI cancer cell lines by virus-mediated introduction of four ES-like genes-c-MYC, SOX2, OCT3/4, and KLF4-followed by a culture more than three months after iPC cells induction. An acquired state was studied by expression of immature-related surface antigens, Tra-1-60, Tra-1-81, Tra-2-49, and Ssea-4; and epigenetic trimethyl modification at lysine 4 of histone H3. Sensitivity to chemotherapeutic agents and tumorigenicity were studied in Lc-iPC cells. RESULTS: Whereas the introduction of defined factors of iPC cells once induced an immature state and sensitized cells to therapeutic reagents, the endogenous expression of the ES-like genes except for activated endogenous c-MYC was down-regulated in a long-term culture, suggesting a high magnitude of the reprogramming induction by defined factors and the requirement of therapeutic maintenance in Lc-iPC cells from cholangiocellular carcinoma HuCC-T1 cells, which harbor TP53(R175H) and KRAS(G12D). The Lc-iPC cells showed resistance to 5-fluorouracil in culture, and high tumorigenic ability with activated endogenous c-MYC in immunodeficient mice. CONCLUSION: The Lc-iPC cells from HuCC-T1 might be prone to an undesirable therapeutic response because of an association with the activated endogenous c-MYC. To consider the possible therapeutic approach in GI cancer, it would be necessary to develop a predictive method for evaluating the improper reprogramming-associated aggressive phenotype of iPC cells.

Modification of sphingoglycolipids and sulfolipids in kidney cell lines under heat stress: activation of monohexosylceramide synthesis as a ceramide scavenger.

Heat stress on Madin-Darby canine kidney cells increased ceramide content to 187% at 40 degrees C for 24 h, and the de novo synthesis from serine increased to 146%. Glucosylceramide (GlcCer) and galactosylceramide (GalCer) synthesis from ceramide, the first glycosylation step of sphingolipid metabolism in kidney cells, increased to 290% (GalCer) and 143% (GlcCer) after metabolic labeling with (14)C-glucose at 42 degrees C for 20 h. The more complex glycolipid lactosylceramide also increased to 151%, whereas sulfatide and ganglioside GM3 decreased to 21% and 43%, respectively. Sulfatide (SM4s) showed optimal sulfation at 37 degrees C, whereas cholesterol sulfate was optimally sulfated at 40 degrees C. The gene expression of ceramide glucosyltransferase (GluT), ceramide galactosyltransferase, and GalCer sulfotransferase (GST) after 24 h culture at 42 degrees C significantly increased to 714%, 221%, and 174%, respectively. Another kidney cell line, COS7, showed less activation of these transferases by heat stress. Although GST gene expression was higher under heat stress, SM4s synthesis decreased, which may have been due to increased GST sensitivity to a temperature higher than 37 degrees C. When we introduced the HSP70 gene into the expression vector and transfected the plasmid (pCDM-dHSP70) into kidney cells, GlcCer synthesis increased significantly. From these results, we speculated that HSP70 may play a role in GluT gene expression to increase GlcCer and decrease intracellular ceramide level.

Development and application of a system for seminolipid metabolism using mouse seminiferous tubules.

A convenient tool for studying metabolism of seminolipid in testis was developed by using mouse isolated seminiferous tubules prepared by collagenase treatment. Because more than 99% of [(35)S]sulfate-incorporation was distributed in seminolipid, its metabolism in seminiferous tubules can be analyzed without disturbance of the other sulfolipids in this assay system. Furthermore, the contents of seminolipid and its precursor, galactosylalkylacylglycerol, which were determined by liquid chromatography-electrospray ionization mass spectrometry, did not change within a few hours, indicating that the incorporations of [(35)S]sulfate into seminolipid solely reflects the turnover rate of this sulfolipid. As an initial application of this system, we characterized heat-susceptibility of the seminolipid turnover rate in mouse seminiferous tubules. Severe heating (44 degrees C for 10 min) of the isolated seminiferous tubules suppressed the (35)S-incorporation into seminolipid to 47% of heating at scrotal temperature (32 degrees C for 70 min). In contrast, pretreatment of the testis in vivo under the same condition (44 degrees C for 10 min) did not decrease the seminolipid turnover rate in the isolated seminiferous tubules. In addition, the activity of galactocerebroside sulfotransferase decreased in the temperature-dependent manner in seminiferous tubules as well as crude tubular homogenates, where the activity is significantly more stable in the former than the latter. The newly developed system could provide useful basic data for further analyses of seminolipid metabolism in the testis.

Defined factors induce reprogramming of gastrointestinal cancer cells.

Although cancer is a disease with genetic and epigenetic origins, the possible effects of reprogramming by defined factors remain to be fully understood. We studied the effects of the induction or inhibition of cancer-related genes and immature status-related genes whose alterations have been reported in gastrointestinal cancer cells. Retroviral-mediated introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins, including Nanog, Ssea4, Tra-1-60, and Tra-1-80 in esophageal, stomach, colorectal, liver, pancreatic, and cholangiocellular cancer cells. Induced cells, but not parental cells, possessed the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, which was supported by epigenetic studies, indicating methylation of DNA strands and the histone H3 protein at lysine 4 in promoter regions of pluripotency-associated genes such as NANOG. In in vitro analysis induced cells showed slow proliferation and were sensitized to differentiation-inducing treatment, and in vivo tumorigenesis was reduced in NOD/SCID mice. This study demonstrated that pluripotency was manifested in induced cells, and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation-inducing treatment. Retroviral-mediated introduction of iPC cells confers higher sensitivity to chemotherapeutic agents and differentiation-inducing treatment.

Epithelial-mesenchymal transition with expression of SNAI1-induced chemoresistance in colorectal cancer.

BACKGROUND: Previous reports have demonstrated that SNAI1 plays a role in epithelial-mesenchymal transition (EMT) through the suppression of CDH1. Its role in the pathology and regulation of EMT expression to chemoresistance in colorectal cancer (CRC) has not yet been fully elucidated. METHODS: Immunohistochemistry was performed to evaluate the expression of Snai1 protein in 30 primary CRC samples. The biological significance of Snai1 expression was studied by induction of the wild-type (WT) and mutant SNAI1 gene in CRC SW480 cells. RESULTS: Examination of 20 surgical specimens of CRC indicated that Snai1 protein expression was localized outer regions of invasive tumors. Introduction of phosphorylation-defective active EMT forms, SNAI1-6SA and SNAI1-8SA, caused downregulation of CDH1 and upregulation of VIM compared with SNAI1-WT and the negative control (NC). Chemoresistance to 5-fluorouracil (IC50) was higher in SNAI1-6SA and SNAI1-8SA transfectants compared with SNAI1-WT and NC. All the above results were significantly different. CONCLUSION: The present study demonstrated that Snai1 plays a role in CRC invasion through phosphorylation, suggesting a plausible mechanism for overcoming chemoresistance that will lead to the development of effective treatments for CRC.

Screening of biomarker genes activated by irradiation of ultraviolet B rays in mouse lymph node M10 cells.

When M10 cells derived from mouse lymph nodes were irradiated with the UVB lamp at a peak emission of 312 nm, the cell growth was suppressed in proportion to irradiation time (10-30 s) and cell apoptosis was also induced by the irradiation. Dynamic changes in 597 genes after exposing these cells to UVB irradiation were investigated by DNA array analysis using array membranes and a (33)P-labeling probe. After 2 h of irradiation, the gene expression in the cells was examined and compared with that in untreated cells. Radioactivity was analyzed using Array Gauge software. The data were further processed using software, EX-ARRAY, which was developed for extracting significant data from the results of 2 background-subtraction methods, i.e., global and local background subtraction. The number of genes suppressed under UV irradiation increased with irradiation time, while that of activated genes decreased. Finally, we confirmed 4 genes (HMG-14, CDX-2, MCP-3, and GRP-78) to be up-regulated and confirmed their activation by northern blot. We propose these genes as the new biomarkers of lymphocyte sensitive to UVB irradiation.

Effect of nutritional substrate on sulfolipids metabolic turnover in isolated renal tubules from rat.

Effects of a glycolytic (glucose) and a gluconeogenic renal nutritional substrate (glutamine) on metabolic turnover of sulfolipids, determined as [(35)S]sulfate incorporation, were compared in renal tubules prepared from well-fed rats. The results showed that the effects of glucose and glutamine, at nearly physiological serum concentration, are quite contrary to each other. Glucose increased the turnover rates of relatively long chain ganglio-series sulfoglycolipids (Gg(3)Cer II(3)-sulfate and Gg(4)Cer II(3),IV(3)-bis-sulfate) (1.7 to 2.4-fold), but not of cholesterol 3-sulfate (0.9-fold). In contrast, glutamine accelerated the turnover rates of relatively short chain sulfoglycolipids (glucosyl sulfatide, galactosyl sulfatide and lactosyl sulfatide) (1.3 to 2.7-fold), as well as cholesterol 3-sulfate (2.4-fold). The possible mechanism which causes these marked differences is also discussed.

Higher expression of renal sulfoglycolipids in marine mammals.

Patterns and contents of major acidic glycosphingolipids in the kidney of three marine mammalian species, the Steller sea lion (Pinnipedia), the rough-toothed dolphin and the broad-beaked dolphin (Odontoceti), were examined, and compared with those of terrestrial mesic mammals. The profile of major acidic glycosphingolipids was not significantly different between the terrestrial and marine mammals: predominant gangliosides were GM3 and GD3, and major sulfoglycolipids were SM4s and SM3. On the other hand, the total concentration (nmol/g wet tissue) of sulfoglycolipids was considerably higher in the marine mammals (2.3-3.0 times) than that in the terrestrial mesic mammals with comparable body weights. In contrast, there was no significant difference in the level of renal glycolipids-bound sialic acid between the marine and the terrestrial mammals. These results suggest that higher expression of renal sulfoglycolipids in marine mammals may contribute to the maintenance of osmotic balance of their body fluid against sea water.

DR negativity is a distinctive feature of M1/M2 AML cases with NPM1 mutation.

Our previous observation of a higher incidence of FLT3-ITD in DR(-) M1/M2 AML than in DR(+) M1/M2 led to an investigation of NPM1 mutation in the same samples, since DR(-) AML and AML with NPM1 mutation share such characteristics as normal karyotype, the absence of CD34, and FLT3-ITD. NPM1 mutation was found in 18 of 26 (69.2%) of DR(-) cases, but not in any of 28 DR(+) cases. FLT3-ITD was noted in 66.7% of the cases with NPM1 mutation. These findings point to DR negativity as another phenotypic feature of AML with NPM1 mutation.

Metabolic responses of sulfatide and related glycolipids in Madin-Darby canine kidney (MDCK) cells under osmotic stresses.

Incorporation of (35)S-sulfate into the polar molecular species of sulfoglycolipids (SM4s) in Madin-Darby canine kidney cells increased in a hypertonic medium (500 mOsm/L) supplemented with sodium chloride. The unknown sulfoglycolipid (SX) was identified as GlcCer sulfate based on the results of TLC, GLC, and mass spectra. The synthesis of SX increased in the hypotonic medium unlike that of SM4s and SM3. TLC showed that hypertonic stress induced the accumulation of GalCer as a precursor of SM4s, whereas hypotonic stress increased GlcCer as a precursor of GlcCer sulfate. The level of ceramide as a precursor of both GalCer and GlcCer increased under hypertonic stress and decreased under hypotonic stress. Cerebroside sulfotransferase mRNA was shown to be elevated in the hyperosmotic condition but not in the hypotonic condition. The increase in SM4s under hypertonic stress was induced by the activation of both the ceramide galactosyltransferase and the cerebroside sulfotransferase genes, whereas the increase in GlcCer sulfate under hypotonic stress was caused by the accumulation of GlcCer as the result of activation of ceramide glucosyltransferase.

HLA-DR-negative AML (M1 and M2): FLT3 mutations (ITD and D835) and cell-surface antigen expression.

FLT3 mutations and cell-surface antigen were investigated in 29 DR-negative (DR(-)) M1/M2 AML samples in comparison with 30 DR-positive (DR(+)) M1/M2 AML samples. FLT3-ITD was detected in 59.3% and D835 was detected in 7.4% of the samples. The incidence of FLT3-ITD was higher in the DR(-) group (59.3%) than in the DR(+) group (17.9%; P=0.002). The DR(-) status was associated with the CD34(-) (82.8%), CD7(-) (92.9%) and CD45RO(+) status (76%). Our results indicated that FLT3 mutation is the most common gene alteration found in the DR(-) M1/M2 AML. These results are important for further characterizing this phenotypic AML entity.

[Radio-frequency ablation therapy for metastatic liver tumors from colorectal carcinoma].

UNLABELLED: Radio-frequency ablation therapy (RFA) as a treatment for metastatic liver tumors from colorectal carcinoma was examined. METHODS: Ten patients with a total of 30 liver metastases from colorectal carcinoma were treated using a Cool-tip RF system from March 2003 to December 2004. RESULTS: Patients had a mean age of 69.8 years and the mean diameter of the metastatic lesions was 29.5 mm (range, 5-82). Two patients had received RFA therapy 2 times, and another 2 patients had received 3 times. Critical complications were not seen, though 5 therapies were performed using CT-guided trans-pulmonary puncture. The rate of partial recurrence was 23.1% and the average observation period was 14.8 months. The partial recurrence had occurred within the mean period of 6.2 months. Although after multimodal therapy was given, it is suggested that repeated RFA for the liver metastasis would improve survival rates. CONCLUSION: RFA is a safe and effective treatment for metastatic liver tumors from colorectal carcinoma as multimodal therapy.

Metabolism of sulfolipids in isolated renal tubules from rat.

Proximal-rich tubules were prepared from rat kidneys by using collagenase treatment. The isolated rat renal tubules were compared with the intact kidney on the following characteristics. (1) Composition of the sulfoglycolipid. (2) Sulfoglycolipid metabolism based on incorporation of [35S]sulfate or some properties of sulfoglycolipid metabolism, including the activities of anabolic and catabolic enzymes. The results indicated following characteristics of the isolated renal tubules in comparison to the kidney in vivo. (1) The sulfoglycolipid compositions are qualitatively similar, except that the content of glucosyl sulfatide, Gg3Cer II3-sulfate, and GM4 was slightly higher in the isolated tubules. (2) The apparent half-lives (15-55 min) of sulfoglycolipids in the isolated tubules could indicate the existence of a rapid turnover pool of these lipids. (3) The sulfotransferase and sulfatase activities related to sulfoamphiphiles in the renal tubule were similar to those reported for the whole kidney. Based on the above criteria, we conclude that the isolated rat renal tubule should be a useful metabolic system for clarification of the short-term physiological events, up to 90 min, of proximal tubular sulfoglycolipids. By using the present system, we showed that biosynthesis of the renal total sulfoglycolipid was significantly elevated in rats deprived of water for 24 h.

[A case of lymph node metastasis after 7 years from Hartmann's operation for stage IV sigmoid colon carcinoma].

The case was a 49-year-old woman. In 1995, she underwent Hartmann's operation for sigmoid colon carcinoma. Histological stage was IV [se, n4(+), P0, H0, M(-)]. Adjuvant chemotherapy was performed by MMC and 5'-DFUR. After 7 years from the surgery, she had a lumbar pain. CT examination revealed a retroperitoneal tumor-like shadow and Para-aortic lymph nodes were swelling. In 2002, she underwent probe laparotomy. There were massive lymph nodes metastasis, and tumor resection was impossible. Following several systemic chemotherapies such as UFT/CPT-11 and TS-1, the tumor progressed and liver metastasis appeared. After 9 years from the first operation, she is still able to eat and her performance status is 1.