Assessment of tumor characteristics based on glycoform analysis of membrane-tethered MUC1.

This corrects the article DOI: 10.1038/labinvest.2017.53.

Assessment of tumor characteristics based on glycoform analysis of membrane-tethered MUC1.

Clinical tissue specimens are useful for pathological diagnosis, which is, in some cases, supported by visualization of biomolecule localization. In general, diagnostic specificity in molecular pathology is increased by the acquisition of a probe to distinguish the modification of isomers. Although glycosylation is one of the candidate modifications in a protein, comparative glycan analysis of disease-associated proteins derived from a single tissue section is still challenging because of the lack of analytical sensitivity. Here we demonstrate a possible method for differential glycoform analysis of an endogenous tumor-associated glycoprotein MUC1 by an antibody-overlay lectin microarray. Tissue sections (5 µm thick) of patients with cholangiocarcinoma (CCA; n=21) and pancreatic ductal adenocarcinoma (PDAC; n=50) were stained with an anti-MUC1 antibody MY.1E12 that was established as a monoclonal antibody recognizing an MUC1 glycosylation isoform with a sialyl-core 1 structure (NeuAcα2-3galactosyl ß1-3-N-acetylgalactosamine). MY.1E12-positive tissue areas (2.5 mm2) were selectively dissected with a laser capture microdissection procedure. The membrane MUC1 was enriched by immunoprecipitation with MY.1E12 and subjected to lectin microarray analysis. Even though the reactivities of MY.1E12 between CCA and PDAC were similar, the lectin-binding patterns varied. We found Maackia amurensis leukoagglutinin and pokeweed lectin distinguished MY.1E12-reactive MUC1 of CCA from that of PDAC. Moreover, MUC1 with M. amurensis hemagglutinin (MAH) reactivity potentially reflected the degree of malignancy. These results were confirmed with MAH-MY.1E12 double fluorescent immunostaining. These glycan changes on MUC1 were detected with high sensitivity owing to the cluster effect of immobilized lectins on a tandem repeat peptide antigen covered with highly dense glycosylation such as mucin. Our approach provides the information to investigate novel glycodynamics in biology, for example, glycoalteration, as well as diseases related to not only MUC1 but also other membrane proteins.

GLI2 is a novel therapeutic target for metastasis of osteosarcoma.

Aberrant activation of the Hedgehog (Hh) pathway has been reported in several malignancies. We previously demonstrated that knockdown of GLI2 inhibited proliferation of osteosarcoma cells through regulation of the cell cycle. In this study, we analyzed the function of GLI2 in the pathogenesis of osteosarcoma metastasis. Immunohistochemical studies showed that GLI2 was overexpressed in patient osteosarcoma specimens. Knockdown of GLI2 inhibited migration and invasion of osteosarcoma cells. In contrast, the forced expression of constitutively active GLI2 in mesenchymal stem cells promoted invasion. In addition, xenograft models showed that knockdown of GLI2 decreased lung metastasis of osteosarcomas. To examine clinical applications, we evaluated the efficacy of arsenic trioxide (ATO), which is a Food and Drug Administration-approved antitumor drug, on osteosarcoma cells. ATO treatment suppressed the invasiveness of osteosarcoma cells by inhibiting the transcriptional activity of GLI2. In addition, the combination of Hh inhibitors including ATO, vismodegib and GANT61 prevented migration and metastasis of osteosarcoma cells. Consequently, our findings suggested that GLI2 regulated metastasis as well as the progression of osteosarcomas. Inhibition of the GLI2 transcription may be an effective therapeutic method for preventing osteosarcoma metastasis.

Combination of MUC1 and MUC4 expression predicts clinical outcome in patients with oral squamous cell carcinoma.

BACKGROUND: Both MUC1 and MUC4 are high molecular weight glycoproteins and are independent indicators of worse prognosis in many human epithelial cancers including oral squamous cell carcinoma (OSCC). However, there has been no investigation of the clinical importance of the co-expression of MUC1 and MUC4 in OSCC. The aim of this study was to evaluate the co-expression profile of MUC1/MUC4 and analyze the prognostic significance in OSCC. METHODS: We examined the expression profile of MUC1 and MUC4 in OSCC tissues from 206 patients using immunohistochemistry. The co-expression profile of MUC1/MUC4 and its prognostic significance in OSCC was statistically analyzed. RESULTS: MUC1 and MUC4 overexpression were strongly correlated with each other (p < 0.0001) and a combination of both MUC1 and MUC4 expression was a powerful indicator for tumor aggressiveness such as tumor size (p = 0.014), lymph node metastasis (0.0001), tumor stage (p = 0.006), diffuse invasion (p = 0.028), and vascular invasion (p = 0.014). The MUC1/MUC4 double-positive patients showed the poorest overall and disease-free survival. Multivariate analysis revealed that MUC1/MUC4 double-positivity was the strong independent prognostic factor for overall and disease-free survival (p = 0.007 and (p = 0.0019), in addition to regional recurrence (p = 0.0025). CONCLUSIONS: Taken together, these observations indicate that the use of a combination of MUC1/MUC4 can predict outcomes for patients with OSCC. This combination is also a useful marker for predicting regional recurrence. MUC1 and MUC4 may be attractive targets for the selection of treatment methods in OSCC.

Two autopsy cases of severe fever with thrombocytopenia syndrome (SFTS) in Japan: a pathognomonic histological feature and unique complication of SFTS.

We report two autopsy cases of severe fever with thrombocytopenia syndrome (SFTS) with a high fatality rate in aged Japanese patients. Both cases were caused by a tick-bite. The pathognomonic histological feature was necrotizing lymphadenitis of systemic lymphoid tissue with SFTS viruses and SFTSV-RNA copies. Marked fungal infections were also observed in the lungs of both patients. Since cellular immune function may be suppressed in SFTS patients, physicians should be aware of possible fungal infections.

Ki-67 is a strong prognostic marker of non-small cell lung cancer when tissue heterogeneity is considered.

BACKGROUND: Ki-67 expression is a well-established prognostic marker in various cancers. However, Ki-67 expression is also known as being heterogeneous. We investigated the prognostic significance of Ki-67 from the view of staining heterogeneity by the technique of Spiral Array. METHODS: 100 cases of resected lung cancer from Toyama university hospital archive were collected. Spiral Array blocks were generated out of 100 cases using 100 µm thick paraffin sections. Four µm thick sections of the Array block were stained for Ki-67. Staining results in each reel were scored for areas with lowest (LS), highest (HS), and average (AS) expression, exclusively in the cancer cells. Heterogeneity score (HeS) was designed as the difference between HS and LS. The scores were divided into four grades (0-3). Clinical information was collected, and the prognostic significance of Ki-67 was analyzed. RESULTS: Pathological stage was available for 91 patients (43 stage IA, 22 stage IB, 2 stage IIA, 9 stage IIB, 13 stage IIIA, 1 stage IIIB, and 1 stage IV). The HS of Ki-67 score in non-small cell lung cancer was 3 in 17 cases, 2 in 27 cases, 1 in 28 cases, 0 in 21 cases, and 4 reels were lost. 78 cases had clinical follow up. 74 cases had all the information available and were analyzed for correlation between Ki-67 expression and survival. Cases with score 2 and 3 of HS and HeS showed significant poorer prognosis (both P < 0.001), whereas LS or AS did not show significance. The results were identical when analyzing adenocarcinoma and squamous cell carcinoma, separately. Cox multivariate analysis of Ki-67 showed that HS was an independent risk factor affecting overall survival. CONCLUSIONS: Ki-67 is a strong prognostic marker for non-small cell lung cancer when the degree of highest staining frequency or heterogeneity is considered.

Castleman's disease in the retroperitoneal space mimicking a paraspinal schwannoma: a case report.

BACKGROUND: Castleman's disease is a rare disease characterized by lymph node hyperplasia. Its occurrence in the retroperitoneal space has rarely been reported, making its preoperative diagnosis difficult. Here, we report a case of retroperitoneal Castleman's disease, which radiologically resembled paraspinal schwannoma. CASE PRESENTATION: A 33-year-old Japanese man with epigastric discomfort underwent abdominal ultrasonic examination revealing a solid mass next to the right kidney. Computed tomography demonstrated a well-circumscribed mass with central calcification in the right psoas muscle. Because the mass presented a dumbbell-like shape extending to the intervertebral foramen, neurogenic tumor was suspected. Both iodine-123 metaiodobenzylguanidine and gallium-67 scintigraphies were negative in the mass, whereas thallium-201 mildly accumulated in the tumor, suggesting blood flow to the tumor. Positron emission tomography revealed accumulation of fluorine-18-2-fluoro-2-deoxy-d-glucose in the tumor at a standard uptake value of 4.7, whereas no other abnormal uptake suggestive of metastatic lesion was noted. On the basis of imaging studies, we mostly suspected paraspinal schwannoma, although malignancy was not completely excluded. Angiography showed feeding vessels from the right lumbar arteries, which were embolized with porous gelatin particles in order to reduce intraoperative bleeding. Surgical resection was performed using a retroperitoneal approach, which revealed the tumor in the swollen psoas muscle. Intraoperative pathological examination of a frozen section revealed no evidence of malignancy; thus, marginal excision of the tumor was performed. The tumor adhered tightly to surrounding muscle tissues, resulting in 940 g of intraoperative blood loss. The pathological examination demonstrated infiltration of lymphocytes surrounding small germinal centers with extensive capillary proliferation. Immunostaining revealed that proliferated lymphocytes were CD3-negative and CD79a-positive. CONCLUSIONS: Although a dumbbell-shaped mass in a paraspinal region is indicative of a schwannoma for orthopedic surgeons, the possibility of Castleman's disease should be considered if a central low-signal area in fissured and a radial pattern is detected on computed tomography or magnetic resonance imaging. Appropriate preparation for massive bleeding during the treatment of Castleman's disease, including angiography and embolization, would be helpful for performing surgical procedures safely.

MUC4: a novel prognostic factor of oral squamous cell carcinoma.

MUC4 mucin is now known to be expressed in various normal and cancer tissues. We have previously reported that MUC4 expression is a novel prognostic factor in several malignant tumors; however, it has not been investigated in oral squamous cell carcinoma (OSCC). The aim of our study is to evaluate the prognostic significance of MUC4 expression in OSCC. We examined the expression profile of MUC4 in OSCC tissues from 150 patients using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed. MUC4 was expressed in 61 of the 150 patients with OSCC. MUC4 expression was significantly correlated with higher T classification (p = 0.0004), positive nodal metastasis (p = 0.049), advanced tumor stage (p = 0.002), diffuse invasion of cancer cells (p = 0.004) and patient's death (p = 0.004) in OSCC. Multivariate analysis showed that MUC4 expression (p = 0.011), tumor location (p = 0.032) and diffuse invasion (p = 0.009) were statistically significant risk factors. Backward stepwise multivariate analysis demonstrated MUC4 expression (p = 0.0015) and diffuse invasion (p = 0.018) to be statistically significant independent risk factors of poor survival in OSCC. The disease-free and overall survival of patients with MUC4 expression was significantly worse than those without MUC4 expression (p < 0.0001 and p = 0.0001). In addition, the MUC4 expression was a significant risk factor for local recurrence and subsequent nodal metastasis in OSCC (p = 0.017 and p = 0.0001). We first report MUC4 overexpression is an independent factor for poor prognosis of patients with OSCC; therefore, patients with OSCC showing positive MUC4 expression should be followed up carefully.

DF3 epitope expression on MUC1 mucin is associated with tumor aggressiveness, subsequent lymph node metastasis, and poor prognosis in patients with oral squamous cell carcinoma.

BACKGROUND: DF3/MUC1 mucin is expressed in various cancer tissues, and many in vitro studies have suggested that it may play a role in the aggressive behavior of malignant tumors. However, to the best of the authors' knowledge, the relation between DF3/MUC1 expression and outcome has not yet been investigated in patients with oral squamous cell carcinoma (OSCC). The objective of the current study was to evaluate the prognostic significance of DF3/MUC1 expression in patients with OSCC. METHODS: The expression profile of DF3/MUC1 in OSCC tissues from 206 patients was examined using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed on the basis of detailed clinicopathologic factors. RESULTS: DF3/MUC1 expression was found to be significantly correlated with tumor aggressiveness, such as pathologic lymph node metastasis (P = .002), advanced tumor stage (P = .02), diffuse invasion of cancer cells (P = .03), and vascular invasion (P = .01). Respectively, the overall survival (OS)and disease-free survival (DFS) rates were significantly worse for patients with DF3/MUC1 expression compared with those without DF3/MUC1 expression (P = .001 and P = .0003, respectively). Multivariate analysis demonstrated that DF3/MUC1 expression was an independent prognostic factor for both OS and DFS (P = .04 for both). In addition, DF3/MUC1 expression was found to be an independent risk factor for subsequent regional lymph node metastasis (P = .03). CONCLUSIONS: Aberrant expression of DF3/MUC1 is an independent prognostic factor indicating poor prognosis in patients with OSCC. DF3/MUC1 expression is a risk factor for subsequent lymph node metastasis in patients with OSCC and therefore may represent an indication for elective neck dissection. Patients with OSCC demonstrating positive expression of DF3/MUC1 should be followed carefully.

Aberrant DNA methylation of tumor-related genes in oral rinse: a noninvasive method for detection of oral squamous cell carcinoma.

BACKGROUND: The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor-related genes with the objective of establishing a noninvasive method for the detection of OSCC. METHODS: Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation-specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene's methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection. RESULTS: Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E-cadherin (ECAD), transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains 2 (TMEFF2), retinoic acid receptor beta (RARß), and O-6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARß, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT. CONCLUSIONS: The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC.

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells.

BACKGROUND: Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. METHODS: Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. RESULT: The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted. CONCLUSIONS: The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

Pathobiological implications of MUC16/CA125 expression in intrahepatic cholangiocarcinoma-mass forming type.

OBJECTIVES: MUC16 carries the peptide epitope CA125, which is well known as a marker of ovarian cancer. High serum levels of MUC16 (CA125) have been reported not only in patients with ovarian cancer but also in patients with liver diseases. We evaluated the expression of MUC16 in intrahepatic cholangiocarcinoma-mass forming type (ICC-MF) tissues. METHODS: We examined the expression of MUC16 by immunohistochemical analyses using the monoclonal antibody M11 in ICC-MF tissues from 63 patients. To compare the prevalence of each mucin expression by clinicopathological features, appropriate statistical analysis was performed. RESULTS: MUC16 was detected in 48% of samples (30/63). After adjusting for the effects of other prognostic factors, multivariate survival analysis revealed that MUC16 expression is a significant independent factor of poor prognosis (p = 0.005). CONCLUSION: The current results indicate that MUC16 expression is a prognostic factor of poor survival in ICC-MF.

A novel anti-MUC1 antibody against the MUC1 cytoplasmic tail domain: use in sensitive identification of poorly differentiated cells in adenocarcinoma of the stomach.

BACKGROUND: Isolated cancer cells of non-solid type poorly differentiated adenocarcinoma (por2) or signet-ring cell carcinoma (sig) are frequently seen in scirrhous gastric cancers with a very poor prognosis. These cells are often scattered in granulation tissue or desmoplastic fibrotic tissue and tend to be overlooked in routine pathological examination. We aimed to raise a novel antibody that can identify the isolated cancer cells easily. METHODS: Because the MUC1 cytoplasmic tail domain (CTD) has many biological roles including tumor progression and cell adhesion disturbance and is expected to be expressed in isolated cancer cells, we raised a novel monoclonal antibody (MAb) MUC1-014E against an intracellular nonrepeating 19-amino-acid sequence (RYVPPSSTDRSPYEKVSAG: N-1217-1235-C) of the MUC1 CTD, using a synthetic peptide including the 7-amino-acid epitope (STDRSPY: N-1223-1229-C). RESULTS: In the immunohistochemical staining of 107 gastrectomy specimens including 48 por2 and 31 sig lesions, the MAb MUC1-014E showed high rates of positive staining (≥5% of carcinoma cells stained) for por2 (100%) and sig (97%), and of the highest intensity staining (4+, ≥75% of carcinoma cells stained) for por2 (100%) and sig (90%). In the 89 biopsy specimens including 82 por2 and 38 sig lesions, the MAb MUC1-014E showed high rates of positive staining for por2 (100%) and sig (100%) and of 4+ staining for por2 (87%) and sig (84%). All the rates were significantly higher than those with cytokeratins (AE1/AE3 or CAM5.2). CONCLUSIONS: The MAb MUC1-014E is very useful for accurate detection of isolated cancer cells in scirrhous gastric cancers.

Comprehensive epigenetic analysis using oral rinse samples: a pilot study.

PURPOSE: To prove that chromatin immunoprecipitation assay can be performed with oral rinse samples and to develop a protocol for comprehensive analysis of functional interactions among DNA methylation, histone modification, and gene expression using such samples. MATERIALS AND METHODS: Eleven cancer cell lines and oral rinse samples from 10 patients with oral squamous cell carcinoma and 3 healthy subjects were examined. The expression of CDKN2A, a tumor suppressor gene, was determined by reverse transcription/polymerase chain reaction and immunohistochemistry. Promoter DNA methylation was assessed by methylation-specific polymerase chain reaction. Chromatin modifications were analyzed by a chromatin immunoprecipitation assay using antibodies for dimethylation and acetylation of lysine 9 of histone H3. RESULTS: Epigenetic control of CDK2NA was observed in vitro in 11 cancer cell lines. Using the present protocol, comprehensive epigenetic analysis could be successfully performed with oral rinse samples. All patients were comfortable using the prescribed amount (16 mL) of normal saline to rinse their mouths. Nine patients (90%) and 1 healthy subject (33%) showed dimethylation of lysine 9 of histone H3. Moreover, 8 patients (80%) showed hypoacetylation of lysine 9 of histone H3, which was not observed in healthy subjects. CONCLUSIONS: The present study showed for the first time that chromatin modifications can be analyzed using oral rinse samples by chromatin immunoprecipitation analysis. To evaluate the contribution of histone modifications for carcinogenesis of oral squamous cell carcinoma, studies including a larger number of subjects should be conducted in the future.

Prognostic relevance of morphological types of intraductal papillary mucinous neoplasms of the pancreas.

OBJECTIVE: The clinicopathological significance of four morphological types of intraductal papillary mucinous neoplasms of the pancreas (IPMNs; gastric, intestinal, pancreatobiliary and oncocytic) was assessed. DESIGN: Retrospective multicentre analysis of 283 surgically resected IPMNs. RESULTS: Of the 283 IPMNs, 139 were of the gastric type, 101 were intestinal, 19 were pancreatobiliary and 24 were oncocytic. These types were significantly associated with clinicopathological factors including sex (p = 0.0032), age (p = 0.00924), ectatic duct size (p = 0.0245), detection of mural nodules (p = 4.09 × 10⁻6), histological grade (p < 2.20 × 10⁻¹6), macroscopic types with differential involvement of the pancreatic duct system (p = 3.91 × 10⁻5), invasive phenotypes (p = 3.34 × 10⁻¹²), stage (p < 2.20 × 10⁻¹6) and recurrence (p = 0.00574). Kaplan-Meier analysis showed significant differences in patient survival by morphological type (p = 5.24 × 10⁻6). Survival rates at 5 and 10 years, respectively, were 0.937 (95% CI 0.892 to 0.984) for patients with gastric-type IPMNs; 0.886 (95% CI 0.813 to 0.965) and 0.685 (95% CI 0.553 to 0.849) for those with intestinal-type IPMNs; 0.839 (95% CI 0.684 to 1.000) and 0.734 (95% CI 0.526 to 1.000) for those with oncocytic-type IPMNs; and 0.520 (95% CI 0.298 to 0.909) and undetermined for those with pancreatobiliary-type IPMNs. Analysis by the Cox proportional hazards model comparing prognostic risks determined by stage and the morphological and macroscopic types indicated that staging was the most significant predictor of survival (p = 3.68×10⁻8) followed by the morphological type (p = 0.0435). Furthermore, the morphological type remained a significant predictor in a subcohort of invasive cases (p = 0.0089). CONCLUSION: In this multicentre retrospective analysis, the morphological type of IPMN appears to be an independent predictor of patient prognosis.

DNA methylation and histone H3-K9 modifications contribute to MUC17 expression.

MUC17 glycoprotein is a membrane-associated mucin that is mainly expressed in the digestive tract. It has been suggested that MUC17 expression is correlated with the malignancy potential of pancreatic ductal adenocarcinomas (PDACs). In the present study, we provided the first report of the MUC17 gene expression through epigenetic regulation such as promoter methylation, histone modification and microRNA (miRNA) expression. Near the transcriptional start site, the DNA methylation level of MUC17-negative cancer cell lines (e.g. PANC1) was high, whereas that of MUC17-positive cells (e.g. AsPC-1) was low. Histone H3-K9 (H3-K9) modification status was also closely related to MUC17 expression. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC17 expression. Furthermore, the hypomethylation status was observed in patients with PDAC. This indicates that the hypomethylation status in the MUC17 promoter could be a novel epigenetic marker for the diagnosis of PDAC. In addition, the result of miRNA microarray analysis showed that five potential miRNA candidates existed. It is also possible that the MUC17 might be post-transcriptionally regulated by miRNA targeting to the 3'-untranslated region of its mRNA. These understandings of the epigenetic changes of MUC17 may be of importance for the diagnosis of carcinogenic risk and the prediction of outcomes for cancer patients.

Human papillomavirus infection in lung and esophageal cancers: analysis of 485 Asian cases.

The role of human papillomavirus (HPV) in the development of lung and esophageal cancer remains inconclusive, which is in contrast to the established role HPV plays in the development of uterine cervical cancer. One of the reasons for this is the difference among reported HPV infection rates in these cancers. An analysis of 485 lung and esophageal cancers (176 lung squamous cell carcinoma, 128 lung adenocarcinoma, 181 esophageal carcinoma) in eight institutions in Asia (Tokyo, Kochi, Kagoshima, and Okinawa, Japan; Seoul and Daegu, Korea; Changhua, Republic of China (Taiwan); Singapore, Singapore) was carried out in order to clarify infection rates with HPV. Samples were examined in one laboratory of the Department of Pathology, the University of Tokyo, Japan in order to avoid inter-laboratory variation using a combination of polymerase chain reaction and in situ hybridization (ISH). HPV was found in 6.3%, 7%, and 9.4% of patients with lung squamous cell carcinoma, lung adenocarcinoma, and esophageal cancer, respectively. Among the geographic areas surveyed, Kagoshima exhibited a significantly higher prevalence of HPV infection in cases of esophageal carcinoma (24.1%). There was no geographical difference in the infection rates of HPV in lung carcinomas. Subtype-specific ISH was also performed, which identified the high-risk HPV types 16/18 in the majority (75.7%) of the patients with lung and esophageal cancer positive for HPV.

Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure.

Mucins are a family of heavily glycosylated high molecular mass proteins that have great potential as novel clinical biomarkers for the diagnosis of various malignant tumors. Supported molecular matrix electrophoresis (SMME) is a new type of membrane electrophoresis that can be used to characterize mucins. In SMME, mucins migrate in a molecular matrix supported by membrane materials. Here, we have developed an immunostaining method for the identification of SMME-separated mucins. The novel method involves stably fixing the mucins onto the SMME membrane and optimizing the molecular matrix for the fixation process. We applied this technique for the detection of MUC1 produced from three cancer cell lines (T47D, HPAF-II and BxPC3) and also analyzed their O-linked glycans by mass spectrometry. Our results revealed that properties of the MUC1 molecules from the three cell lines are different in terms of migrating position in SMME and glycan profile. The present method allows simple and rapid characterization of mucins in terms of both glycans and core proteins. The method will be a useful tool for the exploration of mucin alterations associated with various diseases such as cancer.

Uterine cervical carcinomas associated with lobular endocervical glandular hyperplasia.

AIMS: To investigate the clinicopathological features of cervical uterine carcinoma associated with lobular endocervical glandular hyperplasia (LEGH). METHODS AND RESULTS: Subjects comprised 12 patients with cervical carcinoma associated with LEGH. Carcinoma included nine invasive adenocarcinomas, two adenocarcinoma in-situ (AIS) and one microinvasive squamous cell carcinoma (SCC). Baseline characteristics, cervical cytology, human papilloma virus (HPV) status, immunohistochemistry, surgical procedures and clinical outcomes were investigated. There was a pair of adenocarcinoma cases in a mother and daughter unrelated to Peutz-Jeghers syndrome. In all patients, atypical cells were seen on cervical cytology (adenocarcinoma cells in 11 cases and SCC cells in one). All the 12 patients were positive for mucin antigen 6 (MUC6) in the LEGH component, while seven patients were positive for gastric mucin (HIK1083). HPV was detected only in the SCC component. One patient with adenocarcinoma stage Ib died of disease 4 years after radical hysterectomy. The others are currently alive 2-16 years postoperatively. CONCLUSIONS: The diagnosis of adenocarcinoma with LEGH is not always difficult. The prognosis of adenocarcinoma associated with LEGH may be better than previously expected. Adenocarcinoma with a LEGH component does not always develop into a highly aggressive minimal deviation adenocarcinoma.

Mucins in human neoplasms: clinical pathology, gene expression and diagnostic application.

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis and tumor invasion. Our immunohistochemical studies demonstrated that MUC1 or MUC4 expression is related to the aggressive behavior and poor outcome of human neoplasms. MUC2 is expressed in indolent pancreatobiliary neoplasms, but these tumors sometimes show invasive growth with MUC1 expression in invasive areas. MUC5AC shows de novo high expression in many types of precancerous lesions of pancreatobiliary cancers and is an effective marker for early detection of the neoplasms. The combination of MUC1, MUC2, MUC4 and MUC5AC expression may be useful for early detection and evaluation of the potential for malignancy of pancreatobiliary neoplasms. Regarding the mechanism of mucin expression, we have recently reported that expression of the mucin genes is regulated epigenetically in cancer cell lines, using quantitative MassARRAY analysis, methylation-specific polymerase chain reaction analysis and chromatin immunoprecipitation analysis, with confirmation by the treatment with 5-aza-2'-deoxycytidine and trichostatin A. We have also developed a monoclonal antibody against the MUC1 cytoplasmic tail domain, which has many biological roles. Based on all of the above findings, we suggest that translational research into mucin gene expression mechanisms, including epigenetics, may provide new tools for early and accurate detection of human neoplasms.