Expression and localization of Reg IV in human neoplastic and non-neoplastic tissues: Reg IV expression is associated with intestinal and neuroendocrine differentiation in gastric adenocarcinoma

Regenerating islet-derived family, member 4 (Reg IV) is a candidate marker for cancer and inflammatory bowel disease. In the present study, immunohistochemical analysis of Reg IV was performed in various human neoplastic (n = 289) and non-neoplastic tissues. In the stomach, foveolar epithelium was negative for Reg IV, whereas goblet cells of intestinal metaplasia and neuroendocrine cells at the base of intestinal metaplasia expressed Reg IV. Neuroendocrine cells of the small intestine and colon showed strong expression of Reg IV, whereas goblet cells of the small intestine and colon showed weak or no expression of Reg IV. Insulin-producing beta cells of the endocrine pancreas were positive for Reg IV. Among 143 gastric adenocarcinomas, Reg IV expression was detected in 42 (29.4%) and was associated with both the intestinal mucin phenotype and neuroendocrine differentiation. No association was found between Reg IV expression and clinical characteristics such as tumour stage and patient prognosis. Of 36 colorectal adenocarcinomas, 13 (36.1%) were positive for Reg IV, which was associated with tumour stage (p = 0.0379, Fisher's exact test). Expression of Reg IV was detected in 14 (93.3%) of 15 colorectal carcinoid tumours. Reg IV expression was also detected in 5 (21.7%) of 23 ductal adenocarcinomas of the pancreas. In contrast, lung cancers (n = 30) and breast cancers (n = 30) did not express Reg IV. This is the first immunohistochemical analysis of the expression and distribution of Reg IV protein in human tumours. These data suggest that Reg IV is expressed by gastrointestinal and pancreatic tumours, including adenocarcinomas and carcinoid tumours, and that Reg IV is associated with intestinal and neuroendocrine differentiation of the stomach and gastric carcinoma.     

Diversity in expression and prognostic significance of G1/S cyclins in human primary lung carcinomas

Expression of cyclin A, cyclin E and cdk2 was examined immunohistochemically in 144 cases of primary non-small cell lung carcinoma to evaluate their prognostic value. Cyclin A was co-expressed with cdk2 in the proliferating cells, ie those showing positive Ki-67 staining. The labelling index (LI) of cyclin A revealed a positive correlation with the S-phase fraction and an inverse correlation with histological differentiation. Furthermore, high cyclin A LIs indicated a poor prognosis in all histological types. Cyclin E exhibited a characteristic staining pattern: in squamous cell carcinoma (SCC), differentiated cells without Ki-67 staining revealed cyclin E positivity with expression of cdk2. Conversely, in adenocarcinoma (AC), proliferating cells revealed cyclin E positivity. Cases of large cell carcinoma showed heterogeneous cyclin E staining patterns, unlike those of SCC or AC. Statistical analyses also revealed a marked contrast between SCC and AC. In AC, the LI of cyclin E was inversely correlated with histological differentiation and a high LI predicted a worse prognosis. In contrast, in SCC, the LI of cyclin E correlated positively with histological differentiation and better prognosis. However, the expression levels of cyclin E mRNA evaluated by quantitative RT-PCR were higher in poorly differentiated SCC and AC, suggesting that protein turnover plays a large role in determining cyclin E protein levels. Although the expression of cyclins was demonstrated to be diversely regulated depending on the histological type, the combined immunohistochemical analyses performed in this study on these proteins could be useful tools for evaluating patient prognosis in lung carcinomas.     

Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA-binding protein Musashi-1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi-1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi-1 mRNA expression in the endometrium compared to the myometrium. Musashi-1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi-1 with its molecular target Notch-1 and telomerase. In proliferative endometrium, the proportion of Musashi-1-positive cells in the basalis layer was significantly increased 1.5-fold in the stroma, and three-fold in endometrial glands compared to the functionalis. The number of Musashi-1 expressing cell groups was significantly increased (four-fold) in proliferative compared to secretory endometrium. Musashi-1 expressing stromal cell and cell group numbers were significantly increased (five-fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi-1-expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma.     

CDX2 co-localizes with liver-intestine cadherin in intestinal metaplasia and adenocarcinoma of the stomach

CDX2 and liver-intestine (LI)-cadherin are intestine-specific markers and both are physiologically expressed in the small intestine and colon. Recent studies have demonstrated that CDX2 regulates LI-cadherin gene (CDH17) expression in colorectal cancer. The present study investigated the relationship of CDX2 and LI-cadherin expression in gastric cancer. One hundred and nine pairs of tumour and non-cancerous gastric mucosa were collected from gastrectomy specimens. Protein expression levels of CDX2 and LI-cadherin were determined by immunohistochemical staining. Semi-quantitative RT-PCR showed that the mRNAs of both CDX2 and CDH17 were highly expressed in tumour compared with non-cancerous mucosa. Overexpression of CDX2 was significantly associated with CDH17 in gastric adenocarcinoma. Furthermore, the expression of CDX2 and LI-cadherin proteins was strongly coupled in intestinal metaplasia. In conclusion, overexpression of CDH17 is significantly associated with CDX2.     

PTEN expression is maintained in sporadic colorectal tumours.

Loss of PTEN (phosphatase and tensin homologue deleted from chromosome 10) function has been implicated in the progression of several types of cancer. Allele loss close to the PTEN locus occurs in sporadic colon cancer and germline PTEN mutations cause Cowden disease, an inherited cancer syndrome characterized by an increased incidence of gastrointestinal tract lesions that can progress to colorectal carcinoma. However, although PTEN is a good candidate for involvement in the pathogenesis of sporadic colon cancer, previous analyses have not revealed a high frequency of somatic mutations in colorectal tumours. Alternative mechanisms which could lead to a loss of PTEN expression in colon cancer have not been investigated. This study monitored PTEN mRNA and protein levels in a panel of 50 tumour tissues obtained from 35 patients with sporadic colon cancer. RT-PCR and immunohistochemistry were used to evaluate the expression of mRNA and protein, respectively, in normal, adenoma and adenocarcinoma colorectal tissues as well as in metastatic lesions. To overcome the problem of heterogeneity and normal stromal cell contamination in homogenized tissue specimens, specific cell types were isolated by microdissection prior to PCR analysis. No loss of PTEN expression was evident in any of the colon tissues examined. PTEN protein was localized exclusively in the cytoplasm of normal and tumour cells and no correlation of immunostaining intensity and tumour stage or grade was revealed. As with previous deletion and mutation analyses, the present study suggests that loss of PTEN expression is not prevalent in sporadic colon cancer.     

Significance of histone methyltransferase SETDB1 expression in colon adenocarcinoma

This study investigated the clinical implications of SETDB1 (also known as KMT1E) in human colon adenocarcinoma. Expression levels of SETDB1 proteins were analyzed by immunohistochemistry staining, and tissue microarrays were used to examine expression profiles in human patients. Our results revealed that SETDB1 protein expression was significantly higher in tumor tissue than in normal tissue for the breast, colon, liver, and lung (p < 0.05). Moreover, an analysis with SurvExpress software suggested that elevated expression of SETDB1 mRNA was significantly associated with the overall survival of colon adenocarcinoma patients (p < 0.05); and additional analysis involving 90 paired samples of colon adenocarcinoma tissue and normal tissue revealed that SETDB1 protein expression was 82% higher in cancerous cells (p < 0.001). High SETDB1 expression was also found to be significantly correlated with histological grade (p = 0.005), TNM stage (p = 0.003), T‐class/primary tumor (p = 0.001), and N‐class/regional lymph nodes (p = 0.017); and Kaplan–Meier survival curves indicated that SETDB1 protein expression was significantly associated with poor survival. Finally, univariate analysis demonstrated that SETDB1 protein expression was related to TNM stage (p = 0.004) and SETDB1 score (p = 0.001), whereas multivariate analysis showed that the influence of SETDB1 on overall colon adenocarcinoma survival was independent from other risk factors. Taken together, our results suggest that the SETDB1 protein could serve as a clinical prognostic indicator for colon adenocarcinoma.     

EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival.

One hundred patients with breast carcinoma followed for 7-11 years were included in the present study of EGFR family members, using immunohistochemistry and RT-PCR. By immunohistochemistry, 36%, 27%, 26%, and 82% of the tumours were positive for EGFR, c-erbB-2, c-erbB-3, and c-erbB-4. All the immunoreactive tumours were confirmed positive by RT-PCR. Tumour size, histological grade, lymph node status, S-phase fraction, and stage were confirmed to be significantly associated with both disease-free and cancer-specific survival in the present study. Methods of treatment, histological type, and ploidy had no significant effect on survival. Statistical analysis of EGFR family members in these tumours showed a significant association between c-erbB-2 expression and reduced disease-free and cancer-specific survival. c-erbB-4 expression was associated with a more favourable outcome. Co-expression of c-erbB-2 and EGFR was associated with a worse prognosis. c-erbB-4 expression, however, showed an antagonistic effect on the clinical influence of c-erbB-2 expression. In conclusion, c-erbB-2 expression in breast carcinomas is associated with an unfavourable clinical course and EGFR expression has a synergistic effect. However, c-erbB-4 antagonizes the c-erbB-2 effect on clinical course in breast carcinomas. To achieve best results with immunotherapy against the c-erbB-2 receptor, clarifying the status of c-erbB-4 expression may be of significance.     

Analysis of CBP (CREBBP) gene deletions in Rubinstein-Taybi syndrome patients using real-time quantitative PCR

Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome characterized by facial abnormalities, broad thumbs, broad big toes, and growth and mental retardation as the main clinical features. RTS was shown to be associated with disruption of the CREB-binding protein gene CBP (CREBBP), either by gross chromosomal rearrangements or by point mutations. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (about 10%). Most deletion studies in RTS are performed by FISH analysis, and five cosmids must be used to cover the whole of the CBP gene, which spreads over 150 kb. Here we report the design of gene dosage assays by real-time quantitative PCR that are targeted on three exons located respectively at the 5' end (exon 2), in the middle (exon 12), and at the 3' end (exon 30) of the CBP gene. This technique proved to be efficient and powerful in finding deletions and complementary to the other available techniques, since it allowed us to identify deletions at the 3' end of the gene that had been missed by FISH analysis, and to refine some deletion breakpoints. Our results therefore suggest that real-time quantitative PCR is a useful technique to be included in the deletion search in RTS patients.     

Tumor-to-tumor metastasis to a thyroid follicular adenoma as the initial presentation of a colonic adenocarcinoma

The incidence of thyroid involvement by metastatic disease from distant organs ranges from an average of 3.1% in surgical series to 5.3% in autopsy series. However, the metastasis of one tumor into another (traditionally referred to as 'tumor-to-tumor metastasis') is distinctly uncommon. Typically, they are identified as new manifestations or necropsy findings of a known, pre-existing donor tumor. Herein is described the case of a 59-year-old woman whose thyroid nodule (a follicular adenoma) was resected and found to contain foci of a well-differentiated adenocarcinoma with a morphologic and immunohistochemical profile consistent with origination from the lower gastrointestinal tract. Subsequent diagnostic work-up revealed a sigmoid colon tumor with metastases to the liver. This is, to the authors' knowledge, the first reported example of a colon adenocarcinoma whose initial clinical manifestation was a metastasis to a thyroid neoplasm and only the third reported example of a colonic adenocarcinoma metastatic to a thyroid tumor. In a review of previously reported examples of tumor-to-tumor metastases involving a thyroid neoplasm as the recipient, the following features were present in the majority: (i) multifocality of the metastatic tumor aggregates; (ii) a total lack of, or only minimal amounts of reaction (desmoplastic, inflammatory or myxoid) of the recipient tumor to the metastatic deposits; and (iii) retention of the histopathologic characteristics of the donor tumor in the metastatic deposits. In general, strikingly divergent morphologic features in an otherwise typical thyroid neoplasm should elicit a differential diagnosis that takes into consideration the possibility of metastasis.     

Persistent HIV-1 replication does not explain low levels of T-cell interferon-gamma mRNA and elevated serum NO(2) (-)/NO(3) (-) in patients with stable CD4 T-cell responses to HAART

HIV-1 infected patients adherent to HAART and displaying stable increases in CD4 T-cell counts differ in their control of HIV replication and one might expect this to reflect depressed immune function. The importance of virological control in functional immune reconstitution was investigated in HIV-1 infected patients who maintained high or undetectable plasma HIV RNA levels over 2-4 years on HAART (discordant and complete responders, respectively). Immunocompetence and immune activation were assessed directly ex vivo and after a short period of culture, as HIV replication in cultures from viraemic patients may artificially depress responses. Expression of cytokine (interferon-gamma, interleukin-5) and chemokine receptor (CCR5, CRTH2) mRNA were determined and soluble CD30 and NO(2) (-)/NO(3) (-) were measured in sera. Unstimulated cells from all patients had low levels of IFNgamma mRNA relative to uninfected controls. Discordant responders had more IFNgamma, IL-5 and CCR5 mRNA in mitogen-stimulated PBMC than complete responders, where the difference could be attributed to CD8-T-cells. Serum NO(2) (-)/NO(3) (-) levels were significantly higher in all patients than controls, with no difference between complete and discordant responders. Serum CD30 levels were significantly higher in discordant responders. These data indicate a persistent immune deficit in immune reconstituted patients irrespective of HIV viral load and associate persistent viral replication with lymphocyte activation, probably involving CD8 T-cells.     

Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real-time RT-PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9-fold in total bone marrow cells (p < 0.001) and up to 4-fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD.     

Distribution of Amelotin in Mouse Tooth Development

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT‐PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.