WDR62 overexpression is associated with a poor prognosis in patients with lung adenocarcinoma.

Human WDR62, which is localized in the cytoplasm including the centrosome, is known to be responsible for primary microcephaly; however, the role of WDR62 abnormality in cancers remains largely unknown. In this study, we aimed to reveal the pathological role of WDR62 abnormality in lung adenocarcinoma (LAC). We first examined the WDR62 mRNA expression level of LAC (n = 64) using a QRT-PCR analysis and found that WDR62 mRNA transcripts were significantly overexpressed in LAC (P = 0.0432, Wilcoxon matched pairs test). An immunohistochemical analysis for LAC (n = 237) showed that WDR62 proteins were also significantly overexpressed in LAC (P < 0.0001, Mann-Whitney U test). A Kaplan-Meier analysis demonstrated that patients with LAC who exhibit WDR62 overexpression have a short overall survival (P = 0.0378, log-rank test), and a multivariate analysis revealed that WDR62 overexpression was an independent predictor of a poor survival outcome among LAC patients (hazard ratio, 2.032; 95% confidence interval, 1.071-3.777; P = 0.0305). Next, we examined the functional effect of WDR62 overexpression on the lung cancer cell line H1299. WDR62-overexpressing lung cancer cells exhibited an increase in cell growth. Moreover, the concurrent overexpression of WDR62 and TPX2, a WDR62-interacting protein that is also overexpressed in LAC, induced centrosome amplification in the lung cells. Finally, we disclosed that the concurrent overexpression of WDR62 and TPX2 is common in diverse human cancers, using data from the Cancer Genome Atlas. These results suggested that WDR62 overexpression is associated with a poor prognosis in patients with LAC and leads to an increase in the malignant potential of lung cells.

Reduced expression of the DNA glycosylase gene MUTYH is associated with an increased number of somatic mutations via a reduction in the DNA repair capacity in prostate adenocarcinoma.

8-Hydroxyguanine (8OHG), a major oxidative DNA lesion, is known to accumulate in prostate cancer; however, the status of one of its repair enzymes, MUTYH, in prostate cancer remains to be elucidated. In this study, we showed that the expression levels of MUTYH mRNA and protein were significantly lower in prostate cancer than in non-cancerous prostatic tissue by examining two independent, publicly available databases and by performing an immunohistochemical analysis of prostate cancer specimens obtained at our hospital, respectively. About two-thirds of the prostate cancers exhibited a reduced MUTYH expression. When the effect of reduced MUTYH expression in prostate adenocarcinoma on the somatic mutation load was examined using data from the Cancer Genome Atlas (TCGA) database, the numbers of total somatic mutations and somatic G:C to T:A mutations were significantly higher in the reduced MUTYH expression group than in the other group (P < 0.0001 and P = 0.0013, respectively). To determine the reason why reduced MUTYH expression leads to somatic mutation loads in prostate adenocarcinoma, we compared the DNA repair capacities between PC-3 prostatic cell line derived clones with different MUTYH expression levels. Both the capacities to cleave DNA containing adenine:8OHG mispairs and to suppress mutations caused by 8OHG were significantly lower in prostatic cell lines with lower MUTYH expression than in prostatic cell lines with higher MUTYH expression. These results suggested that reduced MUTYH expression is associated with somatic mutation loads via a reduction in DNA repair capacity in prostate adenocarcinoma. © 2016 Wiley Periodicals, Inc.

Validity of Tissue Microarray by Immunohistochemistry.

BACKGROUND: Tissue microarrays (TMAs) extract designated areas of tissue paraffin blocks in several units that are millimeters in diameter in a cylindrical fashion, array dozens of these tissue specimens, and then re-embed them. Here, a TMA was utilized to analyze renal cell carcinoma (RCC) specimens with anti-FABP7 and anti-Brn2 antibodies. METHODS: Paraffin-embedded specimens from 114 RCC patients were immunostained with anti-FABP7 and anti-Brn2 antibodies to examine the rate of agreement between the staining of TMA grafts compared to conventional tissue slice grafts. The staining area of the tumor was also examined. RESULTS: The positive ratio of anti-FABP7 was 74% and of anti-Brn2 was 57%. The rate of agreement of each antibody was 100% regardless of tumor size before extraction. CONCLUSIONS: Immunostaining of TMA slices might be effective for the analysis of RCC specimens.

TNK2 gene amplification is a novel predictor of a poor prognosis in patients with gastric cancer.

BACKGROUNDS AND OBJECTIVES: We previously examined the amplification status of 10 kinase genes (PIK3CA, EPHB3, TNK2, PTK7, EGFR, MET, ERBB2, HCK, SRC, and AURKA) in gastric cancer (GC). This study aimed to determine the prognostic significance of these gene amplifications in GC. METHODS: A survival analysis was performed for GC patients. Since TNK2 amplification was identified as a prognostic marker in the analysis, we also examined the functional effect of TNK2 overexpression on gastric cells. RESULTS: A Kaplan-Meier analysis showed that the prognosis of patients with GC exhibiting TNK2 or AURKA amplification was significantly poorer than the prognosis of patients with GC without TNK2 or AURKA amplification. A further multivariate analysis revealed that TNK2 amplification was an independent predictor of a poor survival outcome among patients with GC (hazard ratio, 3.668; 95% confidence interval, 1.513-7.968; P = 0.0056). TNK2-overexpressing GC cells showed an increase in cell migration and non-anchored cell growth. Finally, microarray and pathway analyses revealed the aberrant regulation of some cancer-related pathways in TNK2-overexpressing GC cells. CONCLUSIONS: These results suggested that TNK2 amplification is an independent predictor of a poor prognosis in patients with GC and leads to an increase in the malignant potential of GC cells.

RSPO fusion transcripts in colorectal cancer in Japanese population.

R-spondin (RSPO) gene fusions have recently been discovered in a subset of human colorectal cancer (CRC) in the U.S. population; however, whether the fusion is recurrent in CRC arising in patients from the other demographic areas and whether it is specific for CRC remain uncertain. In this study, we examined 75 primary CRCs and 121 primary lung cancers in the Japanese population for EIF3E-RSPO2 and PTPRK-RSPO3 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of EIF3E-RSPO2 and PTPRK-RSPO3 was not detected in any of the lung carcinomas, RSPO fusions were detected in three (4%) of the 75 CRCs. Two CRCs contained EIF3E-RSPO2 fusion transcripts, and another CRC contained PTPRK-RSPO3 fusion transcripts. Interestingly, in one of the two EIF3E-RSPO2 fusion-positive CRCs, a novel fusion variant form of EIF3E-RSPO2 was identified: exon 1 of EIF3E was connected to exon 2 of RSPO2 by a 351-bp insertion. A quantitative RT-PCR analysis revealed that RSPO mRNA expression was upregulated in the three CRCs containing RSPO fusion transcripts, while it was downregulated in nearly all of the other CRCs. An immunohistochemical analysis and a mutational analysis revealed that the RSPO fusion-containing CRC had a CDX2 cell lineage, was positive for mismatch repair protein expression, and had the wild-type APC allele. Finally, the forced expression of RSPO fusion proteins were shown to endow colorectal cells with an increased growth ability. These results suggest that the expression of RSPO fusion transcripts is related to a subset of CRCs arising in the Japanese population.

Visualization of phosphatidylcholine (16:0/16:0) in type II alveolar epithelial cells in the human lung using imaging mass spectrometry.

Imaging mass spectrometry (MS) is an emerging technique that can detect numerous biomolecular distributions in a non-targeting manner. In the present study, we applied a mass imaging modality, mass microscopy, to human lung tissue and identified several molecules including surfactant constituents in a specific structure of the lung alveoli. Four peaks were identified using imaging MS, and the ion at m/z 772.5, in particular, was localized at some spots in the alveolar walls. Using an MS/MS analysis, the ion was identified as phosphatidylcholine (PC)(16:0/16:0), which is the main component of lung surfactant. In a larger magnification of the lung specimen, PC (16:0/16:0) was distributed in a mottled fashion in a section of the lung. Importantly, the distribution of PC (16:0/16:0) was identical to that of anti-SLC34A2 antibody immunoreactivity, which is known to be a specific marker of type II alveolar epithelial cells, in the same section. Our experience suggests that imaging MS has excellent potential in human pathology research.

Modification of Grocott's staining procedure with heat treatment and oxidation by periodic acid for mucormycosis in tissue: a method to detect Mucor spp.

The sensitivity of the Grocott-modified Gomori's methenamine-silver nitrate technique for the detection of fungi is sometimes low, especially for Mucor spp. We modified the Grocott technique by replacing chromic acid with periodic acid in the oxidation step. The use of periodic acid instead of chromic acid enhanced the detectability of Mucor spp. in histopathological sections. Other parameters should be assessed with a high number of cases under different conditions. We propose our protocol as one of the options in practice, especially in cases suspected of Mucor spp. infection.

Complete remission of primary membranous nephropathy following hepatitis E infection.

Primary membranous nephropathy (PMN) is a major cause of nephrotic syndrome in adults. Studies have shown that one-third of PMN cases undergo spontaneous remission, among which are some cases of infection-related complete remission. Herein, we report the case of a 57-year-old man who achieved complete remission of PMN shortly after the onset of acute hepatitis E infection. At the age of 55 years, the patient developed a nephrotic syndrome, and renal biopsy revealed membranous nephropathy, Ehrenreich-Churg stage 1. Treatment with prednisolone (PSL) reduced urinary protein from 7.8 g/gCre to approximately 1 g/gCre but did not lead to complete remission. However, 7 months after starting treatment, he developed an acute hepatitis E infection after consuming wild boar meat. Immediately after the onset of acute hepatitis E, the patient's urinary protein levels decreased to < 0.3 g/gCre. The PSL dose was subsequently reduced and discontinued after 2 years and 8 months, and complete remission was maintained thereafter. We considered that an increase in the number of regulatory T cells (Tregs) caused by acute hepatitis E infection was associated with PMN remission in this patient.

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer.

BACKGROUND: Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. METHODS AND RESULTS: A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. CONCLUSION: These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

Reduced expression of MUTYH with suppressive activity against mutations caused by 8-hydroxyguanine is a novel predictor of a poor prognosis in human gastric cancer.

The MUTYH gene encodes a DNA glycosylase that can initiate the excision repair of adenine mispaired with 8-hydroxyguanine (8OHG) and is responsible for a susceptibility to multiple colorectal adenomas and carcinomas. To determine whether the MUTYH gene is involved in gastric carcinogenesis, we first examined the expression level of MUTYH in gastric cancer. The reduced expression of MUTYH mRNA transcript was detected in both gastric cancer cell lines and primary gastric cancers using qRT-PCR analysis. Immunohistochemical analysis also showed a significant reduction in MUTYH protein expression in gastric cancer, compared with non-cancerous gastric epithelium (immunohistochemical score, 175.5 ± 43.0 versus 281.5 ± 24.8; p < 0.0001). Among the gastric cancers, the MUTYH expression level was significantly associated with the histopathology (p < 0.0001) and the pT stage (p < 0.001). The outcome of patients with gastric cancer exhibiting low MUTYH expression was significantly worse than the outcome of patients with gastric cancer exhibiting high MUTYH expression (p = 0.0007, log-rank test) and a multivariate analysis revealed that reduced MUTYH expression was an independent predictor of a poor survival outcome among the gastric cancer patients (hazard ratio, 1.865; 95% confidence interval, 1.028-3.529; p = 0.0401). We next compared the functional effects of MUTYH on gastric cancer cells, based on their MUTYH expression levels. MUTYH-over-expressing stable clones of the gastric cancer cell line AGS showed: (a) higher DNA cleavage activity towards adenine:8OHG mispair-containing substrates; (b) higher suppressive activity against mutations caused by 8OHG in a supF forward mutation assay; and (c) higher suppressive activity for cellular proliferation than empty vector-transfected AGS clones. These results suggested that MUTYH is a suppressor of mutations caused by 8OHG in gastric cells and that its reduced expression is associated with a poor prognosis in gastric cancer.

Detection of kinase amplifications in gastric cancer archives using fluorescence in situ hybridization.

To test the feasibility of using bacterial artificial chromosomes (BAC) containing kinases for pathological diagnosis using fluorescence in situ hybridization (FISH), 10 BAC probes containing a gene amplified in 5% or more of a pilot cohort were selected from a previous survey using arbitrarily selected BAC clones harboring 100 kinases. In this report, we describe the prevalence and association with the clinicopathological profile of these selected 10 BAC probes in 365 gastric cancer tissues. FISH analyses using these 10 BAC probes containing loci encoding EGFR, ERBB2(HER2), EPHB3, PIK3CA, MET, PTK7, ACK1, STK15, SRC, and HCK showed detectable amplifications in paraffin-embedded tissue in 2.83% to 13.6% of the gastric cancer tissues. Considerable numbers of the cases showed the co-amplification of two or more of the probes that were tested. BAC probes located within a genome neighborhood, such as PIK3CA, EPHB3, and ACK1 at 3q26-29 or HCK, SRC, and STK15 at 20q11-13.1, were often co-amplified in the same cases, but non-random co-amplifications of genes at distant genomic loci were also observed. These findings provide basic information regarding the creation of a strategy for personalizing gastric cancer therapy, especially when using multiple kinase inhibitors.

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.

Aberrant expression and mutation-inducing activity of AID in human lung cancer.

BACKGROUND: Activation-induced cytidine deaminase (AID) is expressed in B lymphocytes and triggers antibody diversification. Recent reports have indicated that the constitutive expression of AID in mice causes not only lymphomas, but also cancers of some organs including the lung, prompting us to investigate the expression and effect of AID on human lung cancer. MATERIALS AND METHODS: We examined AID mRNA expression in 17 lung cancer cell lines and 51 primary lung cancers using a quantitative RT-PCR analysis. Next, we established H1299 lung cancer cells stably overexpressing AID and performed a supF forward mutation assay. We then examined AID protein expression and p53 mutation in 129 primary lung cancers by an immunohistochemical analysis and PCR-SSCP and sequencing analyses, respectively. RESULTS: Aberrant mRNA expression of AID was detected in 29% (5 of 17) of the lung cancer cell lines and 31% (16 of 51) of the primary lung cancers. AID-overexpressing H1299 clones showed a 5.0- to 6.1-fold higher mutation frequency than an empty vector-transfected H1299 clone, and about half of the AID-induced mutations were base substitutions, indicating that AID induces gene mutations in lung cancer cells. Furthermore, an association was found between the AID protein expression level and the p53 mutation status in an analysis of 129 primary lung cancers. A further expression analysis revealed that a portion of AID is localized at the centrosomes. CONCLUSION: Our current findings suggest that the aberrant expression of AID may be involved in a subset of human lung cancers as a result of its mutation-inducing activity.

Allelic imbalances and homozygous deletion on 8p23.2 for stepwise progression of hepatocarcinogenesis.

UNLABELLED: Early hepatocellular carcinoma (eHCC) originates from the hepatocytes of chronic liver disease and develops into classical hepatocellular carcinoma (HCC). To identify sequential genetic changes in multistep hepatocarcinogenesis, we analyzed molecular karyotypes using oligonucleotide genotyping 50K arrays. First, 1q21.3-44 gain and loss of heterozygosity (LOH) on 1p36.21-36.32 and 17p13.1-13.3 were frequently observed in eHCC, but not in chronic liver diseases, suggesting that such chromosomal aberrations are early, possibly causative events in liver cancer. Next, we detected 25 chromosomal loci associated with liver cancer progression in five HCCs with nodule-in-nodule appearance, in which the inner nodule develops within eHCC lesion. Using these chromosomal regions as independent variables, decision tree analysis was applied on 14 early and 25 overt HCCs, and extracted combination of chromosomal gains on 5q11.1-35.3 and 8q11.1-24.3 and LOH on 4q11-34.3 and 8p11.21-23.3 as distinctive attributes, which can classify early and overt HCCs recursively. In these four altered regions identified as late events of hepatocarcinogenesis, two tumors in 32 overt HCCs analyzed in the present study and one in a set of independent samples of 36 overt HCCs in our previous study harbored a homozygous deletion near the CSMD1 locus on 8p23.2. CSMD1 messenger RNA expression was decreased in HCC without 8p23.2 deletion, possibly due to hypermethylation of the CpG islands in its promoter region. CONCLUSION: 1q gain and 1p and 17p LOH are early molecular events, whereas gains in 5q and 8q and LOH on 4q and 8p only occur in advanced HCC, and inactivation of the putative suppressor gene, CSMD1, may be the key event in progression of liver cancer.

Fluorescence in situ hybridization analysis with a tissue microarray: 'FISH and chips' analysis of pathology archives.

Practicing pathologists expect major somatic genetic changes in cancers, because the morphological deviations in the cancers they diagnose are so great that the somatic genetic changes to direct these phenotypes of tumors are supposed to be correspondingly tremendous. Several lines of evidence, especially lines generated by high-throughput genomic sequencing and genome-wide analyses of cancer DNAs are verifying their preoccupations. This article reviews a comprehensive morphological approach to pathology archives that consists of fluorescence in situ hybridization with bacterial artificial chromosome (BAC) probes and screening with tissue microarrays to detect structural changes in chromosomes (copy number alterations and rearrangements) in specimens of human solid tumors. The potential of this approach in the attempt to provide individually tailored medical practice, especially in terms of cancer therapy, is discussed.

Altered expression of the human base excision repair gene NTH1 in gastric cancer.

A base excision repair enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol (Tg), from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against Tg by empty vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced messenger RNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. No associations between clinicopathological factors and NTH1 expression level or localization pattern were detected in the gastric cancers. Next, we found two novel genetic polymorphisms, i.e. c.-163C>G and c.-241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. These findings suggested that downregulation of NTH1 expression and abnormal localization of NTH1 may be involved in the pathogenesis of a subset of gastric cancers.

Copy number estimation algorithms and fluorescence in situ hybridization to describe copy number alterations in human tumors.

The platforms of high-resolution genetic analysis of human tumors have become popular, and several copy number estimation algorithms have been applied to the data generated by single-nucleotide polymorphism microarrays. Although comparisons have been made between several different platforms or methodologies, there has never been a robust comparison of different copy number estimation algorithms, and the validity of the estimations in comparison with multiple fluorescence in situ hybridization (FISH) data in tumors has rarely been addressed. In the present study the dataset that the Affymetrix 250K Nsp array generated in two cancer cases was used to compare the two widely used algorithms for estimating copy number alterations (CNA): the genotyping microarray-based copy number variation (CNV) analysis (GEMCA) algorithm and the copy number analyzer for Affymetrix Genechip mapping (CNAG) algorithm. Considerable differences were noticed between the estimations by these two algorithms, because of the difference in the formula used to calculate the threshold values. Both algorithms yielded highly consistent data with the FISH results, but CNAG was more stringent for detecting loss. There were areas in which both algorithms provided gains, but FISH showed no change. It will be interesting to pursue the reasons for these remaining discrepancies.

Inverse relationship between the length of the EGFR CA repeat polymorphism in lung carcinoma and protein expression of EGFR in the carcinoma.

BACKGROUND AND OBJECTIVES: There is a CA dinucleotide repeat polymorphism in the first intron of the epidermal growth factor receptor (EGFR) gene. Our aim was to examine the relationship between the CA repeat length in lung carcinoma and EGFR mutation, EGFR gene copy number, and EGFR protein expression level in the carcinoma. METHODS: We examined 168 lung carcinomas for the length of the CA repeat polymorphism by PCR-polyacrylamide gel electrophoresis analysis, for EGFR mutations by sequencing analysis, for EGFR gene copy number by fluorescence in situ hybridization analysis, and for EGFR protein expression by immunohistochemical analysis. RESULTS: When the carcinomas were divided into two groups according to the length of their CA repeat polymorphism, the EGFR protein expression level was found to be significantly higher in the shorter allele group than in the longer allele group (P = 0.0116), but its length was not associated with EGFR somatic mutations, high EGFR gene copy numbers, or clinicopathological factors, such as histological type or stage. CONCLUSIONS: The results suggested that the length of the EGFR CA repeat polymorphism in lung carcinoma is inversely related with level of EGFR protein expression in the carcinoma.

Malignant pheochromocytoma in a young adult forming the structure simulating Homer Wright rosette: differentiation from neuroblastoma on repeating fluorescence in situ hybridization.

A peculiar adrenal tumor was analyzed using immunohistochemistry, electron microscopy, and fluorescence in situ hybridization (FISH) with multiple bacterial artificial chromosome (BAC) probes. The patient was a 34-year-old woman with a mass above the left kidney and multiple metastases. Her serum and urine dopamine level were elevated, and a diagnosis of malignant pheochromocytoma was made. The patient died approximately 3 years after her first visit. On post-mortem an adrenal tumor composed of small round cells forming Homer Wright rosette-like structures, a feature rarely observed in pheochromocytoma, was found. Immunohistochemistry was positive for chromogranin A and synaptophysin, and negative for cytokeratin, vimentin and neurofilaments. Because these results did not rule out a diagnosis of neuroblastoma, the tumor was further characterized on FISH with multiple BAC probes for loci known to be altered in neuroblastoma or pheochromocytoma, according to information in the literature that was for the most part obtained using comparative genomic hybridization. FISH demonstrated loss of heterozygosity at 11p, and gains at 16p, 19p, and 19q, a profile that favored a diagnosis of malignant pheochromocytoma over neuroblastoma. This case demonstrates that repeating FISH is useful for differential diagnosis.

Visualization of sphingolipids and phospholipids in the fundic gland mucosa of human stomach using imaging mass spectrometry.

AIM: To analyze the lipid distribution in gastric mucosae. METHODS: Imaging mass spectrometry (MS) is a useful tool to survey the distribution of biomolecules in surgical specimens. Here we used the imaging MS apparatus named iMScope to identify the dominant molecules present in the human gastric mucosa near the fundic glands. Five gastric specimens were subjected to iMScope analysis. These specimens were also analyzed by immunohistochemistry using MUC5AC, H(+)-K(+)-ATPaseß Claudin18 antibodies. RESULTS: Three major molecules with m/z 725.5, 780.5, and 782.5 detected in the gastric mucosa were identified as sphingomyelin (SM) (d18:1/16:0), phosphatidylcholine (PC) (16:0/18:2), and PC (16:0/18:1), respectively, through MS/MS analyses. Using immunohistological staining, SM (d18:1/16:0) signals were mainly co-localized with the foveolar epithelium marker MUC5AC. In contrast, PC (16:0/18:2) signals were observed in the region testing positive for the fundic gland marker H(+)-K(+)-ATPaseß. PC (16:0/18:1) signals were uniformly distributed throughout the mucosa. CONCLUSION: Our basic data will contribute to the studies of lipid species in physical and pathological conditions of the human stomach.