CXCR4+FOXP3+CD25+ lymphocytes accumulate in CXCL12-expressing malignant pleural mesothelioma.

CXCL12 is a chemokine that binds to a G-protein-coupled receptor (CXCR4). CXCL12 is expressed in various tumors and is considered as playing an important role in tumor growth and invasion. The aim of this study is to investigate the expression of CXCL12 in human malignant mesothelioma (MM), the chemotactic effect of CXCL12 derived from MM, and the expression of CXCR4 in MM tissues in relation to regulatory T cells. CXCL12 expression was examined by immunostaining of tissue specimens from malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPEM). The MM group comprised 6 patients (4 men/2 women, MPM=4, MPEM=2, aged 56.0 +/- 12.4 years) and the control (non-mesothelioma) group also had 6 patients (4 men/2 women aged 65.0 +/- 6.7 years). CXCL12 mRNA expression was also examined by RT-PCR in MPM cell lines (H28, H2052, and H2058), while CXCR4 mRNA expression was examined by in situ hybridization in MPM tissue. CXCL12 was expressed in the cytoplasm of MM cells from all patients, but was not expressed in the control group. H2052 and H2058 cells expressed CXCL12 mRNA, but H28 cells did not. CXCL12 in MM tissue homogenate supernatant had a chemotactic effect on CXCR4-expressing THP-1 cells. CXCR4 mRNA was expressed by a part of LCA+CD3+ Foxp3+CD25+ T cells that were located adjacent to the border of CXCL12-expressing epithelioid MPM. These findings suggest that CXCL12 contributed to tumor-related inflammation by inducing the accumulation of CXCR4-expressing cells with regulatory T cell markers around MM.

Cloning of dynein intermediate chain cDNA of the newt Cynops pyrrhogaster.

To isolate genes whose expression is up-regulated after initiation of meiosis, we employed an mRNA differential display method using RNA extracted from newt testis fragments in the spermatogonial and spermatocyte stages. We report here isolation of a spermatocyte stage-specific cDNA clone encoding a newt homologue of dynein intermediate chain (IC). The newt dynein IC cDNA was found to encode a polypeptide consisting of 694 amino acid residues with 66.8% and 45.8% amino acid sequence similarity to sea urchin dynein IC3 and Chlamydomonas IC69, respectively. The predicted protein contains five WD repeats and a novel repeated motif in the C-terminal region. Northern blot analysis revealed that newt dynein IC mRNA was expressed in the spermatocyte and round spermatid stages, suggesting that dynein IC plays a role in formation of flagella as well as in meiotic events.

Immunohistochemical inactivation of p14ARF concomitant with MDM2 overexpression inversely correlates with p53 overexpression in oral squamous cell carcinoma.

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor called p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor designated p14ARF, which arrests cell growth in G1-S and also in G2-M. Whereas inactivation of p16 has been described as a frequent event in various cancers, including oral cancer, the current function of p14ARF is still poorly understood. A physical association between p14ARF and MDM2 blocks MDM2-induced p53 degradation, resulting in increased levels of p53, which in turn leads to cell cycle arrest. The present study immunohistochemically examined the expression of p16 and p14ARF together with pRb, MDM2 and p53 status in a series of oral cancers. The results showed that p14ARF was frequently absent in the oral cancers (15/37, 41%) as was p16 immunostaining. Concomitant immunopositivity for p14ARF and MDM2 overexpression was frequently observed in a subset of the cancers, whereas an inverse correlation between p14ARF and MDM2 expression and the diffuse staining of p53 was clearly detected. Moreover, the results showed that in most cases of oral cancer (35/37, 95%) at least one protein was altered, and lymph node metastasis was more frequent in the tumors with alterations in both the p16/pRb and p14ARF/p53 pathway (8/16, 50%) than in the tumors with one or no alteration of these two major pathways.

Newt RAD51: cloning of cDNA and analysis of gene expression during spermatogenesis.

A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated. The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95-96% amino acid identity to Xenopus and mammalian RAD51. Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain. In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (> or =eight cells in a cyst), reaching a maximum level in leptotene-zygotene spermatocytes, and thereafter declined. These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.

Clinical significance of immunohistochemical Bcl-2 expression in invasive breast carcinoma.

The immunohistochemical expression of bcl-2 protein, and its correlations with clinicopathological features and prognosis were studied in patients with invasive breast carcinoma. Bcl-2 positive expression significantly correlated with hormone receptor positivity and histological tumor differentiation, and inversely correlated with p53 overaccumulation. No correlation was observed between bcl-2 expression and patient age, menopausal status, tumor size, and lymph node metastasis. Survivals of stage I to III patients who had not received adjuvant hormonal therapy showed no difference between bcl-2-positive and -negative tumors, even if patients were divided as with or without adjuvant chemotherapy, or with or without nodal involvement. In consequence, immunohistochemical bcl-2-positivity correlates with positive hormone receptors and well differentiated phenotypes in invasive breast carcinoma, however, it might not predict response to adjuvant chemotherapy and not be a favorable predictive value in patients treated without adjuvant hormonal therapy.

Granular Cell Tumor of the Breast Diagnosed by Core Needle Biopsy: A Case Report.

Granular cell tumor rarely occurs in the breast. We report a 69-year-old woman with a right breast mass that simulated carcinoma on palpation, mammography, and ultrasonography. Aspiration biopsy cytology showed no malignant atypical cells. Core needle biopsy was performed to obtain an accurate diagnosis. The lesion was histologically confirmed to be a granular cell tumor. Immunostaining was positive for S-100 protein and vimentin, and negative for keratin, carcinoembryonicantigen, estrogen receptor and gross cystic disease fluid protein-15. The tumorwas treated by wide local excision. Surgeons should be aware that granular cell tumor can resemble breast cancer in order to avoid performing a needless radical mastectomy.

In situ hybridization with biotinylated tyramide amplification: detection of human papillomavirus DNA in cervical neoplastic lesions.

A novel peroxidase-mediated amplification system, which is based on the deposition of biotinylated tyramide (BT) molecules, was recently described for immunohistochemistry and in situ hybridization (ISH). We report here a highly sensitive ISH system combined with a microwave prehybridization treatment and BT enhancement after hybridization. Our ISH with BT amplification can detect human papillomavirus (HPV) 16 DNA signals in the fixed SiHa cell, suggesting the ability to detect single or few copies of HPV DNA. To confirm its diagnostic usefulness, we used our ISH with BT amplification to detect HPV 16 DNA in 81 cases of cervical neoplastic lesions, which had been used for routine pathologic diagnosis. Of 81 tumors, 47 (58%) showed HPV 16 DNA, of which 29 and 18 were cervical intraepithelial neoplasia (CINs) and invasive cancers, respectively. In almost all of the invasive cancers positive for HPV 16 DNA, ISH revealed a basic dot signal pattern in the nuclei, suggesting that HPV DNA integrated into tumor cell DNA. On the other hand, low-grade CINs displayed a diffuse, mainly episomal, signal pattern, which decreased in frequency with increased grade of CIN. Our ISH with BT amplification is highly sensitive and can be used to detect various genes and their expressions.

Spindle cell carcinoma of the breast.

Spindle cell carcinoma is a rare breast tumor. We present herein three cases of spindle cell carcinoma of the breast and review its characteristics from the literature. Spindle cell carcinoma frequently forms a large and well-circumscribed tumor with gross cyst formation. Histologically, its dominant component is of sheets of spindle shaped cells, and it includes such contiguous carcinoma components as squamous differentiation or invasive ductal carcinoma. Estrogen receptor expression and lymph node metastasis tend to be low. Despite the sarcomatous features, spindle cells are likely to be derived from epithelial cells of mammary glands. Immunohistochemical and ultrastructural examination demonstrated the expression of keratin and the desmosome-like junctional structure in the spindle cell components. Relatively favorable prognosis is expected in spindle cell carcinoma of the breast compared to common breast carcinoma.

Immunohistochemical expression of metallothionein in invasive breast cancer in relation to proliferative activity, histology and prognosis.

Immunohistochemically detected metallothionein expression [MT(+)] was shown to be related to aggressive behavior of the invasive ductal carcinoma of the breast. In this study, MT expression was examined immunohistochemically in 92 cases of invasive breast carcinoma and compared with immunohistochemically demonstrated estrogen receptor (ER), c-erbB-2, Ki-67 status and clinicopathological characteristics. Of the 92 cases examined, 27.1% (25 cases) were MT(+), and high percentages of the solid tubular subtype of invasive ductal carcinoma (47%), medullary carcinoma (80%), and carcinomas with spindle cell metaplasia (100%) were positive for MT. MT(+) carcinomas showed tendency to have highly atypical nuclei, and nuclear staining for Ki-67 antigen was found in a higher percentage of cases than in MT(-) carcinomas. An inverse relationship between MT(+) and ER immunoreactivity was observed. MT expression was not associated with age distribution, menopausal status, tumor size or lymph node metastasis. The overall survival rate in MT(+) cases was worse than in those negative for MT, but no significant association was found. MT(+) was not associated with poor prognosis in total, estrogen receptor-negative or node-negative tumors. These findings suggest that MT expression in breast cancer cells is related to cell-proliferative activity, and that dedifferentiation of carcinoma cells may play a role in induction of MT expression.

Differential expression of annexin V during spermatogenesis in the newt Cynops pyrrhogaster.

In order to isolate genes whose expression is up-regulated after the initiation of meiosis, we screened a cDNA expression library of newt testes with antiserum against homogenates of testes derived from the spermatogonial and spermatocyte stages. We report the isolation of spermatocyte-specific cDNA clones encoding a newt homologue of the calcium-dependent phospholipid-binding protein, annexin V. Northern blot analysis showed that newt annexin V mRNA was 1.7 kb in length and was expressed strongly in testes, but weakly in other organs. In situ hybridization revealed that the expression of newt annexin mRNA was barely observed in spermatogonia, but increased significantly in leptotene-zygotene primary spermatocytes and reached a maximum level in pachytene spermatocytes and round spermatids. The newt annexin V cDNA predicted a 323-amino acid protein and had a 68% homology to human annexin V. The predicted amino acid sequence contained a conserved 4-fold internal repeat of approximately 70 residues like other annexin proteins. Immunoblot analysis using the monoclonal antibody against newt annexin V showed that the protein was expressed scarcely in spermatogonia but was abundantly expressed in stages from primary spermatocytes to spermatids; this pattern was consistent to that of the mRNA. Immunohistochemical analysis revealed that newt annexin V was localized in the cytoplasm of the spermatogenic cells, but not in somatic cells such as Sertoli cells or pericystic cells. These results indicate that the expression of newt annexin V is up-regulated in the spermatogenic cells after the initiation of meiosis and suggest that newt annexin V plays an important role in spermatogenesis.

Malignant rhabdoid tumor of the breast: a case report.

A 66-year-old woman developed a malignant rhabdoid tumor of the breast, with a tumor doubling time of 10 days. One month after radical mastectomy, there was local recurrence, followed by multiple pulmonary metastases, and the patient died of respiratory failure 5 months after surgery. The gray-white-colored tumor measured 13 x 12 x 10 cm, and its border was well defined. The tumor was composed of diffusely growing round or polygonal cells with vesicular nuclei, prominent nucleoli, and ample cytoplasm containing eosinophilic inclusions. Lymph node involvement was widespread. Both vimentin and keratin were clearly demonstrated by immunohistochemical staining. Ultrastructural studies revealed that the MRT cells contained cytoplasmic whorls of intermediate filaments.