Insertionally polymorphic sites of human endogenous retrovirus-K (HML-2) with long target site duplications.

BACKGROUND: Human endogenous retroviruses (HERVs) belong to the LTR-retrotransposon family, where the complete HERV sequence contains two long terminal repeats (LTRs) located at each end. Intact LTRs possess highly conserved transcriptional promoter and enhancer sequences, so analyses of HERV insertional polymorphisms are expected to provide greater insights into human genomic variation compared with the conventional analysis of single nucleotide variations. High-throughput sequencing technology is developing but genome-wide investigations of HERVs are methodically challenging, and thus a comprehensive understanding of HERV insertional polymorphisms and target site duplications (TSDs) remains elusive. RESULTS: We identified five human-specific insertionally polymorphic sites in HERVK (HML-2), one of the HERV subgroups, by extracting HML-2-deleted sequences from the genomic structural variation database, which we successfully characterized and then updated the existing catalogue of HML-2 insertional polymorphisms. The insertionally polymorphic states were confirmed in a small Japanese population by genomic PCR analysis for four of the five sites identified. Sequencing of the preintegration sites clearly showed that the HML-2 site located at 7p21.2 had 250-base pair (bp) TSDs, which is one of the longest TSDs in HML-2. In addition to these five sites, another insertionally polymorphic site for a non-human-specific HML-2 site was also identified at 6p25.2, which was flanked by 111-bp TSDs and the corresponding ERV locus was also annotated in the genome of non-human primates. CONCLUSIONS: Our analysis demonstrated the existence of HERV insertions flanked by unconventionally long TSDs, including those with lengths as high as 250 bp. This suggests that the length range of retroviral TSDs is larger than considered previously, which might help to understand how retroviral integration occurs in the host genome.

Immunohistochemical expression analysis of leucine-rich PPR-motif-containing protein (LRPPRC), a candidate colorectal cancer biomarker identified by shotgun proteomics using iTRAQ.

BACKGROUND: Colorectal cancer (CRC) is the fourth most frequent cause of cancer deaths in the world. Novel biomarkers for the diagnosis, prognosis, and treatment of CRC are required to improve the clinical strategy. METHODS: We applied shotgun proteomics using isobaric tags for relative and absolute quantitation (iTRAQ) to identify novel biomarkers of CRC, and then we detected leucine-rich PPR-motif-containing protein (LRPPRC) expression in 83 normal colorectal tissues and 133 CRC tissues by immunohistochemistry. RESULTS: A total of 570 proteins were identified using iTRAQ. We validated the expression of LRPPRC protein by immunohistochemical analysis of the 77 proteins that showed expression changes in the cancer tissues >1.5-fold the levels in the normal tissues. The expression levels of LRPPRC were significantly higher in CRC tissues than those in normal colorectal tissues, and the expression levels were related with tumor differentiation and especially high in moderately differentiated CRC tissues. CONCLUSION: We identified a novel, differentially expressed protein, LRPPRC, which has the potential to serve as a molecular target for diagnosis and/or prognosis of CRC.

Prognostic impact of CD73 and A2A adenosine receptor expression in non-small-cell lung cancer.

In immune cells, CD73 dephosphorylates and converts extracellular AMP into adenosine, which binds the A2A adenosine receptor (A2AR). Blockade of this interaction, which induces an immunosuppressed niche in the tumor microenvironment, represents a potential novel treatment strategy. The clinical significance of CD73 and A2AR expression in non-small-cell lung cancer (NSCLC), however, has yet to be thoroughly investigated. Here we evaluated CD73 and A2AR protein expression levels using immunohistochemistry in tissue microarrays containing 642 resected NSCLC specimens. Furthermore, we compared the expression profiles of 133 paired primary tumors and lymph node metastases. CD73 and A2AR expression levels were significantly higher in females than in males, in never smokers than in ever smokers, and in adenocarcinomas than in squamous cell carcinomas. Among adenocarcinomas, significantly higher CD73 and A2AR expression was observed in TTF-1-positive and mutant EGFR-positive tumors than in their counterparts. Compared with CD73, A2AR expression was more inconsistent between primary tumors and lymph node metastases. Among NSCLC patients, high CD73 expression was an independent indicator of poor prognosis in multivariate Cox regression analyses for overall survival [hazard ratio (HR), 2.18; 95% confidence interval (CI), 1.38-3.46] and recurrence-free survival (HR, 2.05; 95% CI, 1.42-2.95). In contrast, high A2AR expression was an independent predictor of favorable prognosis for overall survival (HR, 0.70; 95% CI, 0.50-0.98) and recurrence-free survival (HR, 0.74; 95% CI, 0.56-0.97). Together, these findings indicate that CD73 and A2AR have opposing prognostic effects, although cases involving CD73 or A2AR expression share some clinicopathological features.

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.

Clinical significance of PD-L1 and PD-L2 copy number gains in non-small-cell lung cancer.

New reliable biomarkers are needed to predict the response to immune checkpoint inhibitors against programmed death-1 (PD-1) and its ligand (PD-L1), because PD-L1 expression on tumor cells has limited power for selecting patients who may benefit from such therapy. Here we investigated the significance of PD-L1 and PD-L2 gene copy number gains using fluorescence in situ hybridization as well as PD-L1 and PD-L2 expression in 654 patients with resected non-small-cell lung cancer. The prevalence of PD-L1 amplification and polysomy was 3.1% and 13.2%, respectively. The PD-L1 gene copy number status was in agreement with both the PD-L2 and Janus kinase 2 gene copy number statuses. PD-L1 and PD-L2 expression was observed in 30.7% and 13.1%, respectively. Both PD-L1 copy number gains and expression were associated with smoking-related tumors. Tumor cells with PD-L1 genomic gains exhibited significantly higher levels of PD-L1 expression than those without, but PD-L2 copy number gains were not related to PD-L2 augmentation. PD-L1 gene amplification and polysomy were independently associated with PD-L1 expression, with high immune infiltrates and EGFR expression in a multivariate logistic regression model. Comparative analysis between primary tumors and synchronous regional lymph node metastases revealed that the PD-L1 gene copy number alterations were highly consistent and reproducible compared with the PD-L1 expression. Both PD-L1 amplification and level of protein expression were predictors of poor survival using Cox univariate analyses. Therefore, we conclude that an increase in PD-L1 gene copy number can be a feasible alternative biomarker for predicting response to anti-PD-1/PD-L1 therapy.

Clinicopathological and Survival Analysis of Japanese Patients with Resected Non-Small-Cell Lung Cancer Harboring NKX2-1, SETDB1, MET, HER2, SOX2, FGFR1, or PIK3CA Gene Amplification.

INTRODUCTION: Gene amplification is an important genetic change in cancer cells. We investigated the prevalence, clinicopathological characteristics, and prognostic value of NKX2-1 (also known as TTF-1), SETDB1, MET, HER2, SOX2, FGFR1, and PIK3CA amplification in Japanese patients with non-small-cell lung cancer (NSCLC). METHODS: The copy numbers of the seven above-mentioned genes were assessed using fluorescence in situ hybridization in a tissue microarray containing 282 surgically resected NSCLC specimens (164 adenocarcinoma [AC], 99 squamous cell carcinoma [SCC], and 19 others). Clinicopathological information were obtained from the medical records. RESULTS: NKX2-1, SETDB1, MET, HER2, SOX2, FGFR1, and PIK3CA gene amplification were observed in 30 of 277 (10.8%), 16 of 280 (5.7%), 38 of 278 (13.7%), 8 of 270 (3.0%), 34 of 278 (12.2%), 18 of 282 (6.4%), and 53 of 278 (19.1%) cases, respectively. Coamplification was detected in 16 of 156 (10.3%) AC patients and 35 of 93 (37.6%) SCC patients (p < 0.0001). NKX2-1 amplification was significantly related to an AC histology (p = 0.004), whereas SOX2, FGFR1, and PIK3CA amplifications were related to a SCC histology (p < 0.0001). Within the ACs, NKX2-1 and SETDB1 amplifications were markers of a shorter survival period. A multivariate Cox proportional hazards model revealed that NKX2-1 amplification was an independent predictor of poor survival (hazard ratio, 2.938; 95% confidence interval, 1.434-6.022; p = 0.003). Coamplification had impact on patient outcome in AC but not in entire NSCLC and SCC. CONCLUSIONS: The amplification status differed among the histological types of NSCLC. NKX2-1 amplification was an independent and the most practically important predictor of a poor prognosis among Japanese patients with AC.

SGF29 and Sry pathway in hepatocarcinogenesis.

Deregulated c-Myc expression is a hallmark of many human cancers. We have recently identified a role of mammalian homolog of yeast SPT-ADA-GCN5-acetyltransferas (SAGA) complex component, SAGA-associated factor 29 (SGF29), in regulating the c-Myc overexpression. Here, we discuss the molecular nature of SFG29 in SPT3-TAF9-GCN5-acetyltransferase complex, a counterpart of yeast SAGA complex, and the mechanism through which the elevated SGF29 expression contribute to oncogenic potential of c-Myc in hepatocellularcarcinoma (HCC). We propose that the upstream regulation of SGF29 elicited by sex-determining region Y (Sry) is also augmented in HCC. We hypothesize that c-Myc elevation driven by the deregulated Sry and SGF29 pathway is implicated in the male specific acquisition of human HCCs.

Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation.

Herein, we report the characterization of Limb expression 1-like, (LIX1L), a putative RNA-binding protein (RBP) containing a double-stranded RNA binding motif, which is highly expressed in various cancer tissues. Analysis of MALDI-TOF/TOF mass spectrometry and RNA immunoprecipitation-sequencing of interacting proteins and the microRNAs (miRNAs) bound to LIX1L revealed that LIX1L interacts with proteins (RIOK1, nucleolin and PABPC4) and miRNAs (has-miRNA-520a-5p, -300, -216b, -326, -190a, -548b-3p, -7-5p and -1296) in HEK-293 cells. Moreover, the reduction of phosphorylated Tyr(136) (pTyr(136)) in LIX1L through the homeodomain peptide, PY136, inhibited LIX1L-induced cell proliferation in vitro, and PY136 inhibited MKN45 cell proliferation in vivo. We also determined the miRNA-targeted genes and showed that was apoptosis induced through the reduction of pTyr(136). Moreover, ROS1, HCK, ABL1, ABL2, JAK3, LCK and TYR03 were identified as candidate kinases responsible for the phosphorylation of Tyr(136) of LIX1L. These data provide novel insights into the biological significance of LIX1L, suggesting that this protein might be an RBP, with implications for therapeutic approaches for targeting LIX1L in LIX1L-expressing cancer cells.

SASS6 overexpression is associated with mitotic chromosomal abnormalities and a poor prognosis in patients with colorectal cancer.

Spindle assembly abnormal protein 6 homolog (SASS6) plays an important role in the regulation of centriole duplication. To date, the genetic alteration of SASS6 has not been reported in human cancers. In the present study, we examined whether SASS6 expression is abnormally regulated in colorectal cancers (CRCs). Increased SASS6 mRNA and protein expression levels were observed in 49 (60.5%) of the 81 primary CRCs and 11 (57.9%) of the 19 primary CRCs, respectively. Moreover, the upregulation of SASS6 mRNA expression was statistically significant (P=0.0410). Next, using DLD-1 colon cancer cells inducibly expressing SASS6, SASS6 overexpression was shown to induce centrosome amplification, mitotic abnormalities such as chromosomal misalignment and lagging chromosome, and chromosomal numerical changes. Furthermore, SASS6 overexpression was associated with anaphase bridge formation, a type of mitotic structural abnormality, in primary CRCs (P<0.01). SASS6 upregulation in colon cancer was also revealed in the Cancer Genome Atlas (TCGA) data and was shown to be an independent predictor of poor survival (multivariate analysis: hazard ratio, 2.805; 95% confidence interval, 1.244­7.512; P=0.0112). Finally, further analysis of the TCGA data demonstrated SASS6 upregulation in a modest manner in 8 of 11 cancer types other than colon cancer, and SASS6 upregulation was found to be associated with a poor survival outcome in patients with kidney renal cell carcinoma and lung adenocarcinoma. Our present findings revealed that the upregulation of SASS6 expression is involved in the pathogenesis of CRC and is associated with a poor prognosis among patients with colon cancer. They also suggest that SASS6 upregulation is a genetic abnormality relatively common in human cancer.

NSD3-NUT-expressing midline carcinoma of the lung: first characterization of primary cancer tissue.

BACKGROUND: Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare, aggressive malignancy. Only two pediatric and three adult cases of pulmonary NMCs have been documented. In more than two-thirds of NMC cases, a gene fusion between NUT and BRD4 or BRD3 has been documented; other fusions are rare. CASE PRESENTATION: A 36-year-old woman was admitted because of a rapidly progressing tumor of the lung with metastases to the breast and bone. A biopsy from the lung tumor revealed an undifferentiated neoplasm exhibiting round to oval nuclei with vesicular chromatin, prominent nucleoli, and scant cytoplasm. Immunohistochemical staining demonstrated focal EMA, cytokeratin AE1/AE3, cytokeratin CAM 5.2, p63, CD138, and vimentin positivity. Finally, the nuclear staining pattern for NUT confirmed a histopathological diagnosis of NMC. A 5'- rapid amplification of the cDNA end (RACE) procedure successfully identified the partner of the NUT translocation as NSD3, a recently discovered partner. Fluorescence in situ hybridization confirmed the NSD3-NUT gene rearrangement, whereas a BRD3/4-NUT fusion gene was not detected. CONCLUSION: We herein describe the first case of an NSD3-NUT-expressing NMC of the lung. The further accumulation of variant NMCs should provide clues to the establishment of new individualized therapy for NMCs.

Robust quantitative assessments of cytosine modifications and changes in the expressions of related enzymes in gastric cancer.

BACKGROUND: The rediscovery of 5-hydroxymethylcytosine, the ten-eleven translocation (TET) family, thymine-DNA glycosylase (TDG) and isocitrate dehydrogenase (IDH) have opened new avenues in the study of DNA demethylation pathways in gastric cancer (GC). We performed a comprehensive and robust analysis of these genes and modified cytosines in gastric cancer. METHODS: Liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) was used to assess 5-methyldeoxycytidine (5-mC), 5-hydroxymethyldeoxycytidine (5-hmC), 5-formyldeoxycytidine (5-fC) and 5-carboxyldeoxycytidine (5-caC) quantitatively in tumorous and non-tumorous regions of GCs; [D2]-5-hmC was used as an internal standard. Expression levels of the genes TET1, TET2, TET3, TDG, IDH1 and IDH2 were measured using a real-time reverse transcription polymerase chain reaction (RT-PCR) and were compared to the clinical attributes of each case. Using HEK293T cells the effects of introducing plasmids containing full-length TET1, TET2, and TET3 and 7 variants of the TET2 catalytic domain were evaluated in terms of their effect on cytosine demethylation. RESULTS: LC-MS/MS showed that 5-hmC was significantly decreased in tumorous portions. 5-mC was also moderately decreased in tumors, while 5-fC and 5-caC were barely detectable. The expressions of TET1, TET2, TET3, TDG and IDH2, but not IDH1, were notably decreased in GCs, compared with the adjacent non-tumor portion. TET1 expression and the 5-hmC levels determined using LC-MS/MS had a significantly positive correlation and TET1 protein had a greater effect on the increase in 5-hmC than TET2 and TET3 in HEK293T cells. CONCLUSIONS: The loss of 5-hmC and the down-regulation of TET1-3, TDG and IDH2 were found in GCs. The loss of 5-hmC in GCs was mainly correlated with the down-regulation of TET1.

Single-cell time-of-flight secondary ion mass spectrometry reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells.

Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.

Extensive goblet cell metaplasia of the peripheral lung may harbor precancerous molecular changes: comparison of two cases.

We present two cases of extensive goblet cell metaplasia in the peripheral lung. The first case was a 72-year-old male with a nodule (12 × 10 mm) detected by CT. Macroscopically, it contained abundant mucin; histopathologically, goblet cells were predominant, whereas ciliated and basal cells were sparse. A KRAS G12V mutation was detected. In the pulmonary background of this case, scattered tiny foci of goblet cell metaplasia were present. The second case was a 71-year-old female with a nodule (7 × 5 mm) detected by CT. It contained abundant mucin, and microscopically, various cell types were intermingled. Although the nodule mainly comprised goblet cells, ciliated and basal cells were also easily identified. No KRAS mutation was found in this patient. Dispersed minute foci of goblet cell metaplasia were identified in the pulmonary background. Therefore, we suspect that airway irritants may have contributed to the changes that occurred in the pulmonary background of both cases. We propose that the tendency of goblet cells to overwhelm other cell types in the metaplastic epithelium may be an indicator of precancerous molecular changes.

PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer.

Polo-like kinase 4 (PLK4) is a centrosomal protein that is involved in the regulation of centrosome duplication. This study aimed to determine whether the genetic abnormality of PLK4 is involved in human gastric cancer. First, we examined the status of PLK4 mRNA expression in 7 gastric cancer cell lines and 48 primary gastric cancers using an RT-PCR analysis. The upregulation of PLK4 mRNA expression was detected in 57.1 % (4/7) of the gastric cancer cell lines, and a novel PLK4 variant with exon 4, but without exon 5, was identified. In the primary gastric cancers, the upregulation of PLK4 mRNA expression in the cancerous cells was detected in 50.0 % (24/48) of the cases, and this upregulation was statistically significant (P value = 0.0139). Next, we established AGS gastric cancer cells capable of inducibly expressing PLK4 using the piggyBac transposon vector system and showed that PLK4 overexpression induced centrosome amplification and chromosome instability using immunofluorescence and FISH analyses, respectively. Furthermore, PLK4 overexpression suppressed primary cilia formation. Our current findings suggested that PLK4 is upregulated in a subset of primary gastric cancers and that PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.

A rare Japanese case with a NUT midline carcinoma in the nasal cavity: a case report with immunohistochemical and genetic analyses.

BACKGROUND: NUT (nuclear protein in testis) midline carcinoma (NMC) is a recently described aggressive malignancy that is genetically defined by rearrangements of the NUT locus at 15q14. In approximately two-thirds of cases, the characteristic t(15;19) results in the fusion oncogene BRD4-NUT. Only 10 sinonasal NMCs have been documented, none of which were Japanese cases. CASE PRESENTATION: An 18-year-old woman was admitted because of a rapidly progressing tumor in the nasal cavity. A biopsy revealed an undifferentiated neoplasm without squamous differentiation. The tumor cells had round to oval nuclei with vesicular chromatin, prominent nucleoli, and scant cytoplasm. Immunohistochemical staining demonstrated a strong positivity for vimentin and NUT, with focal CD138 and only spotty EMA and cytokeratin AE1/AE3 staining. Cytogenetic and fluorescence in situ hybridization analyses revealed a t(15;19) and BRD4-NUT gene rearrangement. Direct sequencing identified the in-frame fusion of exon11 of BRD4 with exon2 of NUT. The patient was transferred to another hospital for chemoradiotherapy. CONCLUSION: We herein describe the first Japanese case with an NMC of the sinonasal cavity, providing detailed and unambiguous cyto- and molecular genetic information on BRD4-NUT-rearrangement. The accumulation of cases with well-documented genetic data should provide clues to the treatment of this tumor entity.

Identification and association study with lung cancer for novel insertion polymorphisms of human endogenous retrovirus.

Sequences of human endogenous retroviruses (HERVs) are members of the long terminal repeat (LTR) retrotransposon family. Although the expression of HERV has long been a topic of investigation, HERV-insertion polymorphisms are not well known, and a genetic association between HERV-insertion polymorphisms and cancer has never been reported. To identify novel HERV loci in the genome from cancer tissues, we carried out the inverse PCR method targeting a conserved LTR region of HML-2, which is the most recently acquired HERV group. Novel two insertions, HML-2_sLTR(1p13.2) and HML-2_sLTR(19q12), were identified as insertionally polymorphic solo LTRs. Furthermore, a significant prevalence of HML-2_sLTR(1p13.2) homozygosity was detected in female never-smoking patients aged 60 years and over who had lung adenocarcinoma [versus the other genotyping; odds ratio (OR): 1.97; 95% confidence interval (CI): 1.01-3.81]. In another cohort consisting of female never-smoking patients with lung adenocarcinoma, a prevalence of HML-2_sLTR(1p13.2) homozygosity tended to be high in patients aged 60 years and over (versus the other genotyping; OR: 2.03; 95% CI: 0.96-4.29), whereas a low prevalence of HML-2_sLTR(1p13.2) homozygosity was detected in patients <60 years old (versus the other genotyping; OR: 0.31; 95% CI: 0.11-0.94). Our results suggest that HML-2_sLTR(1p13.2) is involved with the susceptibility to lung adenocarcinoma in female never-smokers in an age-dependent manner and that other HERV polymorphisms related to human diseases might remain to be identified in the human genome.

Accumulated phosphatidylcholine (16:0/16:1) in human colorectal cancer; possible involvement of LPCAT4.

The identification of cancer biomarkers is critical for target-linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non-neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0/16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0/16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0/16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0/16:1) to lyso-PC(16:0) was higher in CRC, we postulated that lyso-PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC(16:0/16:1) in CRC. In an immunohistochemical analysis, LPCAT4 was shown to be overexpressed in CRC. These data indicate the potential usefulness of PC(16:0/16:1) for the clinical diagnosis of CRC and implicate LPCAT4 in the elevated expression of PC(16:0/16:1) in CRC.

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.

Lipid peroxidation-induced DNA adducts in human gastric mucosa.

DNA adducts are a major cause of DNA mutation and DNA mutation-related diseases, but the simultaneous identification of multiple DNA adducts has been a challenge for a decade. An adductome approach using consecutive liquid chromatography and double mass spectrometry after micrococcal nuclease treatment has paved the way to demonstrations of numerous DNA adducts in a single experiment and is expected to contribute to the comprehensive understanding of overall environmental and endogenous exposures to possible mutagens in individuals. In this report, we applied an adductome approach to gastric mucosa samples taken at the time of a gastrectomy for gastric cancer in Lujiang, China, and in Hamamatsu, Japan. Seven lipid peroxidation-related DNA adducts [1,N6-etheno-2'-deoxyadenosine, butanone-etheno-2'-deoxycytidine (BεdC), butanone-etheno-2'-deoxy-5-methylcytidine, butanone-etheno-2'-deoxyadenosine (BεdA), heptanone-etheno-2'-deoxycytidine, heptanone-etheno-2'-deoxyadenosine (HεdA) and heptanone-etheno- 2'-deoxyguanosine] were identified in a total of 22 gastric mucosa samples. The levels of these adducts ranged from 0 to 30,000 per 10(9) bases. Although the presence of Helicobacter pylori DNA in the mucosa was not related to these adducts level, the levels of BεdC, BεdA and HεdA were higher in the Japanese gastric mucosa samples. The profiles of these 7 adduct levels among the 21 cases were capable of discriminating between the possible origins (China or Japan) of the gastric mucosa samples. Our report is the first demonstration of lipid peroxidation-related DNA adducts in the human stomach, and these observations warrant further investigation in the context of the significance of DNA adducts in human gastric carcinogenesis.

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.