Vimentin Immunohistochemistry Research Articles

Radiofrequency preserves histoarchitecture and enhances collagen synthesis in experimental tendon injury.

We investigated the action of radiofrequency (RF) on the healing process after inducing experimental lesions of the Achilles tendon in rats. Wistar rats were surgically subjected to bilateral partial transverse sectioning of the Achilles tendon. The right tendon was treated with radiofrequency (RFT), whereas the left tendon served as a control (CT). On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each, which were treated with monopolar radiofrequency (Tonederm™) until they were sacrificed on the 7th, 14th or 28th days. The histological specimens were studied for inflammatory cell content, collagen types I and III, immunostaining and morphometry. Total collagen were biochemically analyzed and to evalute fibroblast and myofibroblast proliferation by vimentin and α-actin smooth muscle immunohistochemistry methods. Statistical analysis was performed using the Student's t-test, the sign test and the Kruskal-Wallis test to compare tendons treated with radiofrequency with the non-treated tendons (α=5%; α=10%). Larger amounts of collagen I with hydroxyproline content and myofibroblast cells were clearly evident within 7 days (p<0.05). No difference was observed in the inflammatory cell content between the groups. We found better collagen arrangement with RF administration across the entire time studied. Radiofrequency administration preserves histoarchitecture and enhances collagen synthesis during the initial phases of cicatrization, suggesting that the treatment can provide improved stiffness during the most vulnerable phases of tendon healing. Clinical studies may include RF among the therapeutic tools in tendinous lesion management.

Abstract 3924: Establishment and characterization of two cell lines of anaplastic pancreatic cancer

Abstract Background: Anaplastic pancreatic cancer is a rare malignancy with a poor prognosis. As a whole, pancreatic cancer is a devastating prognosis but its therapeutic strategy has not still well developed. Thus, new models are needed to research its biology. Purpose: We have established and characterized 2 anaplastic cancer cell lines (OCUP-A1 and OCUP-A2) for revealing tumor worsening factors and the mechanism of dedifferentiation. Methods: We researched the present studies by comparing with established 2 cell lines and well-known pancreatic cancer cell lines. In vitro proliferation, migration and invasion assays was examined in each cell lines under normoxia and hypoxia by CellPlayer™ 96-Well Kinetic Cell Proliferation, Migration and Invasion Assays. Furthermore,as assessment of chemosensitivity, IC50 values against 5Fu and gemcitabine ware measured by MTT assay. As In vivo assays, the growth of the xenografts in each cell lines was also measured for a month. The immunohistochemistry (IHC) of E-cadherin and vimentin for primary tumors and the xenografts was performed to confirm the induction of epitherial-mesenchymal transition (EMT). Result: Both OCUP-A1 and OCUP-A2 were pleomorphic cells derived from ascites of anaplastic pancreatic cancer patients. The doubling time of OCUP-A1 and OCUP-A2 was 30.9 h and 20.4 h, respectively. In migration and invasion assays, relative wound density in new 2 cell lines ranged from 47.1 % to 98.1 % at 48 hours. The values were not so different from that of other cell lines. Interestingly, although a large part of OCUP-A1 is spindle shape in normoxia, the subpopulation with polygonal structure has increased in hypoxia. Furthermore, OCUP-A1 rapidly proliferated in hypoxia. The IC50 values of gemcitabine for OCUP-A1 and OCUP-A2 were 8.77 nM and 4.90 nM, respectively. And the values of 5-Fu for OCUP-A1 and OCUP-A2 were 48.5 μM and 14.8μM, respectively. Chemosensitivity of 2 established cell lines was not superior to other cell lines. The xenografts of OCUP-A1 and OCUP-A2 more rapidly increased than other cell lines (MIAPaCa2 and Panc-1). In IHC, the E-cadherin expression was reduced and the vimentin expression was enhanced in both specimens. Conclusion: We established 2 anaplastic pancreatic cancer cell lines. It was suspected that these cell lines would have aggressive characters affected by TME and EMT. Citation Format: Kotaro Miura, Kenjiro Kimura, Ryosuke Amano, Sadaaki Yamazoe, Keiichiro Hirata, Masatsune Shibutani, Katsunobu Sakurai, Hisashi Nagahara, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Masakazu Yashiro, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Establishment and characterization of two cell lines of anaplastic pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3924. doi:10.1158/1538-7445.AM2014-3924

CASE REPORT: SPLENIC FIBROSARCOMA IN A CAMPBELL'S HAMSTER

A 2.5-year-old intact female hamster (Phodopus campbelli) was referred to the VMTH of NCHU on May 7th, 2013 for a swelling of left abdomen and significant decrease in appetite. Physical examination revealed the enlarged abdomen slightly affected the patient's mobility. A firm mass was palpated in the left abdominal cavity. Tentative diagnosis was an abdominal mass. An exploratory laparotomy and splenectomy was performed together with the removal of a mass of 1 × 1 × 0.8 cm in size and 3.6 g in weight. Grossly, the mass was well capsulated and covered by a sac which filled with blood-like discharge. Histologically, diagnoses were made according to the results of hematoxylin and eosin, Masson's trichrome and vimentin immunohistochemistry stained sections. Based on the histological characteristics, the final diagnosis was a splenic fibrosarcoma in a Campbell's hamster. The patient is fully recovered after surgery but the hamster died of natural causes four months later.

Qualitative and Quantitative Requirements for Assessing Prognostic Markers in Prostate Cancer.

Molecular prognostic markers are urgently needed in order to improve therapy decisions in prostate cancer. To better understand the requirements for biomarker studies, we re-analyzed prostate cancer tissue microarray immunohistochemistry (IHC) data from 39 prognosis markers in subsets of 50 – >10,000 tumors. We found a strong association between the “prognostic power” of individual markers and the number of tissues that should be minimally included in such studies. The prognostic relevance of more than 90% of the 39 IHC markers could be detected if ≥6400 tissue samples were analyzed. Studying markers of tissue quality, including immunohistochemistry of ets-related gene (ERG) and vimentin, and fluorescence in-situ hybridization analysis of human epidermal growth factor receptor 2 (HER2), we found that 18% of tissues in our tissue microarray (TMA) showed signs of reduced tissue preservation and limited immunoreactivity. Comparing the results of Kaplan-Meier survival analyses or associations to ERG immunohistochemistry in subsets of tumors with and without exclusion of these defective tissues did not reveal statistically relevant differences. In summary, our study demonstrates that TMA-based marker validation studies using biochemical recurrence as an endpoint require at least 6400 individual tissue samples for establishing statistically relevant associations between the expression of molecular markers and patient outcome if weak to moderate prognosticators should also be reliably identified.

Epithelial-to-mesenchymal transition predicts prognosis of pancreatic cancer

Pancreatic cancer has a dismal prognosis that is attributed to common local invasiveness and metastasis. Epithelial-to-mesenchymal transition (EMT) plays an important role in cancer invasion and metastasis and is associated with early dissemination. The aim of this study was to evaluate the association between EMT and the prognoses for patients with pancreatic cancer. Immunohistochemistry of E-cadherin and vimentin was performed on surgical specimens from 174 patients who underwent resection of their pancreatic cancers. Tumoral stainings of E-cadherin and vimentin were graded, and EMT statuses were determined by calculating the ratio of vimentin to E-cadherin, whereby patients were categorized into 3 groups: epithelial, intermediate, and mesenchymal. The correlations between EMT statuses and clinicopathologic factors and prognoses were analyzed. There was a significant correlation between EMT status and CA19-9 levels (P = .020); peritoneal washing cytology (P = .025); portal vein invasion (P = .038); and lymph node metastasis (P = .030). The median survival for patients with epithelial tumors was 40.2 months as compared to 13.7 months for patients with mesenchymal tumors. Multivariate analysis demonstrated that perineural invasion (P = .024); lymph node metastasis (P = .033); and EMT status (P < .0001) were significant prognostic factors. It is interesting that adjuvant chemotherapy (gemcitabine and/or S-1) improved the median survival time from 10.8 to 16.1 months in patients with mesenchymal tumors (P = .002); however, no significant difference was seen in patients with epithelial tumors. EMT status is an important prognostic factor for pancreatic cancer and is associated with portal vein invasion and lymph node metastasis.

Differential remodeling of cadherins and intermediate cytoskeletal filaments influence microenvironment of solid and ascitic sarcoma

Different forms of sarcoma (solid or ascitic) often pose a critical medical situation for pediatric or adolescent group of patients. To date, predisposed genetic anomalies and related changes in protein expression are thought to be responsible for sarcoma development. However, in spite of genetic abnormality, role of tumor microenvironment is also indispensable for the evolving neoplasm. In our present study, we characterized the deferentially remodeled microenvironment in solid and ascitic tumors by sequential immunohistochemistry and flowcytometric analysis of E-cdaherin, N-cadherin, vimentin, and cytokeratin along with angiogenesis and metastasis. In addition, we considered flowcytometric apoptosis and CD133 positive cancer stem cell analysis. Comparative hemogram was also considered as a part. Our investigation revealed that both types of tumor promoted neovascularization over time with sign of local inflammation. Invasion of neighboring skeletal muscle by solid sarcoma was more frequent than its ascitic counterpart. In contrary, rapid and earlier cadherin switching (E-cadherin to N-cadherin) in ascitic sarcoma made them more aggressive than that of solid sarcoma and helped to early metastasize distant tissue like liver through the hematogenous route. Differential cadherin switching and infidelity of cytokeratin expression in Vimentin positive sarcoma also influenced the behavior of ascitic CD133+ cancer initiating cell pool with respect to CD133+ cells housed in solid sarcoma. Therefore our study concludes that differential cadherin switching program and infidelity of intermediate filaments in part, sharply discriminate the severity and metastatic potentiality of either type of sarcoma accompanying with CD133+ cellular repertoire. Besides, tumor phenotype-based dichotomous cadherin switching program could be exploited as a future drug target to manage decompensated malignant ascitic and solid sarcoma.

Epithelial to mesenchymal markers and clinical outcomes on erlotinib in stage IV non-small cell lung cancer patients.

e19117 Background: An epithelial phenotype in NSCLC is associated with improved sensitivity to EGFR tyrosine kinase inhibitors (TKI). The best method to identify this subset is unknown (Richardson Anticancer Research 2012, Byers Clin Cancer Res 2012). This retrospective study correlates E-cadherin (Ecad) and vimentin (vim) immunohistochemistry (IHC) expression with outcomes in advanced NSCLC patients (pts) treated with erlotinib (E). Methods: Advanced NSCLC pts that received E were included if sufficient tumor was available from diagnosis. IHC scores for E-cad and vim were generated by multiplying frequency (0-4) by intensity (0-4). Log Rank was used to correlate IHC expression with progression free and overall survival (PFS, OS). Results were compared to a subset of pts with tissue from primary surgical NSCLC resection who later received E for recurrent disease. Results: 159 advanced NSCLC pts treated with E had tissue from diagnosis and IHC analysis. There was no correlation with PFS or OS on E and high/low vim or Ecad expression. Subtracting the IHC scores (vim minus ecad) created a difference score. A low difference score (n = 62) correlated with prolonged PFS (2.6 vs 1.9 months, p = .014 HR 1.52) compared with a high score, n = 97. Low difference score trended toward prolonged OS (p=.46) 33 of the patients had tissue available from primary surgical resection. The invasive front was examined for membranous E-cad and cytoplasmic vim (Allred score 0-8). Patients with low vim (&lt; 4) and Ecad (&gt;5), n= 19, trended toward prolonged PFS and OS on E compared with patients with high vim (&gt;5) and low Ecad (&lt;6), n=10 (4.2 vs 1.6 months and 15.5 vs 6.5 months, respectively, p=NS). Conclusions: In this retrospective analysis, using unselected, frequently small tissue specimens, the expression of ecad or vim alone by IHC did not correlate with outcomes for E treated patients. A complicated difference score (vimentin score minus ecadherin score) did correlate with PFS on E. Examining EMT markers at the invasive edge of resected NSCLC tumors might more accurately assess EMT activity and its relationship to outcomes when these pts are recommended EGFR-TKIs.

Clinical Experience of Stewart-Treves Syndrome in the Lower Leg

Clinical Experience of Stewart-Treves Syndrome in the Lower Leg

Epithelial-to-Mesenchymal Transition Leads to Docetaxel Resistance in Prostate Cancer and Is Mediated by Reduced Expression of miR-200c and miR-205

Docetaxel is a standard chemotherapy for patients with metastatic prostate cancer. However, the response is rather limited and not all of the patients benefit from this treatment. To uncover key mechanisms of docetaxel insensitivity in prostate cancer, we have established docetaxel-resistant sublines. In this study, we report that docetaxel-resistant cells underwent an epithelial-to-mesenchymal transition during the selection process, leading to diminished E-cadherin levels and up-regulation of mesenchymal markers. Screening for key regulators of an epithelial phenotype revealed a significantly reduced expression of microRNA (miR)-200c and miR-205 in docetaxel-resistant cells. Transfection of either microRNA (miRNA) resulted in re-expression of E-cadherin. Functional assays confirmed reduced adhesive and increased invasive and migratory abilities. Furthermore, we detected an increased subpopulation with stem cell-like properties in resistant cells. Tissue microarray analysis revealed a reduced E-cadherin expression in tumors after neoadjuvant chemotherapy. Low E-cadherin levels could be linked to tumor relapse. The present study uncovers epithelial-to-mesenchymal transition as a hallmark of docetaxel resistance. Therefore, we suggest that this mechanism is at least in part responsible for chemotherapy failure, with implications for the development of novel therapeutics.

Cell Adhesion Molecule Expression in Human Lens Epithelial Cells After Corticosteroid Exposure

Aim: The aim of the study was to investigate changes in cell adhesion molecule expression in human lens epithelial cells (HLEC) subjected to glucocorticoids.Methods: Human lens epithelial cells were exposed to different concentrations of dexamethasone for 24 hours. Cell adhesion molecule expression was studied by western blot and immunohistochemistry of vimentin, N-cadherin, E-cadherin, α-catenin, β-catenin and γ-catenin. Expression of the glucocorticoid receptor (GR) was also studied. Cell morphology was examined by transmission electron microscopy (TEM).Result:Expression of N-cadherin, α-catenin, β-catenin and GR was significantly decreased in dexamethasone exposed cells as compared to unexposed cells. No significant change in γ-catenin was present. Visualization of adhesion molecules, N-cadherin and α-catenin, by immunohistochemistry showed decreased antigen reactivity in dexamethasone exposed as compared to the unexposed cells. However, no change was seen for β-catenin and γ-catenin. E-cadherin was not detectable using western blot or immunohistochemistry.TEM showed multilayering of cells, vacuole formation and appearance of electron-dense multivesicular bodies in HLEC exposed to 0, 0.1, 1, 10 and 100 αM dexamethasone.Conclusion:Glucocorticoids affect several adhesion molecules in lens epithelial cells, something that may contribute to the pathogenesis of posterior subcapsular opacification.

Effect of heterogeneity of HER2 expression on brain metastases of breast cancer.

635 Background: HER2-overexpressing or triple-negative [ER(-)/PR(-)/HER2(-)] breast cancers are associated with increased risk of brain metastases. The mechanisms leading to metastasis in each subtype are not well known. Methods: We introduced the wild-type HER2 gene into MDA-MB-231-luc-D3H2LN (231-Luc) cells, which are triple-negative breast cancer cells, and established HER2-expressing (63.2%) cells as 231-Luc-HER2 cells. We investigated the tumor formation following orthotopic inoculation and brain metastasis following intracardiac injection into nude mice. Metastasis was detected by bioluminescence imaging and confirmed in H&amp;E staining and immunohistochemistry of vimentin and HER2 expressions. Flow cytometry analysis was used to detect the proportion of CD44+/CD24- cells, a maker for stem-like breast cancer cells. Results: 231-Luc-HER2 cells formed larger tumors in orthotopic xenograft models compared to 231-Luc cells, however, no significant difference was observed in proliferation in vitro. Neither 231-Luc-HER2 nor 231-Luc metastasized in the brain from the breast after orthotopic inoculation. After intracardiac injections of the 231-Luc-HER2 cells, brain metastasis developed (7/13 mice, 53.8%). Immunohistochemical analysis revealed that most metastasized cells expressed HER2, although we had injected a mixture of HER2-positive and HER2-negative cancer cells. Interestingly, administering Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, effectively prevented HER2-positive cells to colonize the brain. However, the HER2-negative 231-Luc-HER2 cells developed into brain metastases. In fact, the 231-Luc cells, which are HER2-negative, also metastasized in the brain (10/16 mice, 62.5%). Flow cytometry analysis of the 231-Luc-HER2 cells showed that HER2-positive cells decreased the population of CD44+/CD24- (HER2+/CD44+/CD24-: 86.8% and HER2-/CD44+/CD24-: 96.3%). Conclusions: The mechanism of brain metastases of HER2-positive breast cancer cells is different from that of HER2-negative breast cancer cells. It is therefore important to consider an additional therapeutic approach when dealing with HER2-negative cells in tumors having the heterogeneity of HER2 expression.

Abstract 458: Implication of epithelial to mesenchymal transition (EMT) in pancreatic cancer prognosis

Abstract [Background] In recent years, epithelial-to-mesenchymal transition (EMT) has been shown to play an important role during cancer invasion and metastasis, and is now being actively studied in various cancers. Moreover, EMT is thought to be involved in resistance to chemotherapy and this elucidation could contribute to improved therapeutic effect. A thorough examination of EMT in pancreatic cancer, especially using resected surgical specimens, has not been conducted to date. [Methods] Between April 2004 and March 2010, 174 patients who underwent the resection of their pancreatic cancers were used for this study. Immunohistochemistry of E-cadherin and Vimentin were performed on their resected specimens, and the results of the staining were graded as follows; grade 1: negative, grade 2: positive, grade 3: strong positive. V/E ratio (Vimentin grade/E-cadherin grade) was determined and the patients were categorized into three group as follows: Epithelial(E)1. The correlation between EMT status and clinicopathological factors were analyzed. [Results] 1. Of the 174 cases, EMT status was determined as 37 Epithelial, 48 Borderline and 89 Mesenchymal. We found a significant correlation between EMT status and washing cytology (P = 0.025), tumor size (P = 0.014), portal vein invasion (P = 0.038) and lymphatic invasion (P = 0.047). 2. The median survival was 40.2 months in E, 63.1 months in B, and 13.7 months in M, respectively. There was a significant difference between M and E, and M and B (P &amp;lt; .0001). 3. According to univariate analysis, tumor location (head), CA19-9 level, tumor size (≥ 2cm), anterior tumor invasion, bile duct invasion, duodenal invasion, portal vein invasion, lymphatic or vascular invasion, positive perineural invasion, lymph node metastasis and EMT status (B · M / E) were found to be significant prognostic factors. Multivariate analysis showed perineural invasion (P = 0.024), lymph node metastasis (P = 0.033) and EMT status (P &amp;lt;0.0001) were significant prognostic factors. 4. Adjuvant chemotherapy (gemcitabine or S-1) improved the median survival from 19.9 to 63.1 months in group B, from 10.8 to 16.1 months in group M (P = 0.034, P = 0.002, respectively), however, no significant difference was seen in group E. [Discussion] EMT status was found to be one of the most important prognostic factors in pancreatic cancer and suspected to be associated with cancer invasion and metastasis. Further studies are needed to validate these results, but chemotherapy strategies targeted to epithelial and mesenchymal tumors might improve the outcomes of this disease by increasing the effect of chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 458. doi:1538-7445.AM2012-458

Direct inhibition of the transforming growth factor‐β pathway by protein‐bound polysaccharide through inactivation of Smad2 signaling

Transforming growth factor-β (TGF-β) is involved in the regulation of cell proliferation, differentiation, and apoptosis and is associated with epithelial-mesenchymal transition (EMT). Inhibition of the TGF-β pathway is an attractive strategy for the treatment of cancer. We recently screened for novel TGF-β inhibitors among commercially available drugs and identified protein-bound polysaccharide (PSK) as a strong inhibitor of the TGF-β-induced reporter activity of 3TP-lux, a TGF-β1-responsive luciferase reporter. Protein-bound polysaccharide is used as a non-specific immunostimulant for the treatment of gastric and colorectal cancers in Japan. The anticancer activity of this agent may involve direct regulation of growth factor production and enzyme activity in tumors in addition to its immunomodulatory effect. Although several clinical studies have shown the beneficial therapeutic effects of PSK on various types of tumors, its mechanism of action is not clear. In the present study, Western blot analysis showed that PSK suppressed the phosphorylation and nuclear localization of the Smad2 protein, thereby suggesting that PSK inhibits the Smad and MAPK pathways. Quantitative PCR analysis showed that PSK decreased the expression of several TGF-β pathway target genes. E-cadherin and vimentin immunohistochemistry showed that PSK suppressed TGF-β1-induced EMT, and FACS analysis showed that PSK inhibited the EMT-mediated generation of CD44(+) /CD24(-) cells. These data provide new insights into the mechanisms mediating the TGF-β-inhibiting activity of PSK and suggest that PSK can effectively treat diseases associated with TGF-β signaling.

Cervical carcinoma-associated fibroblasts are DNA diploid and do not show evidence for somatic genetic alterations

BackgroundCancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression.MethodsWe combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) in cervical cancer-associated stromal cell fractions (n = 57) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to confirm LOH findings.ResultsSmooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. However, we found no evidence for DNA aneuploidy, somatic genetic alterations in the vimentin-positive stromal cell fractions of any samples, while high frequencies of DNA content abnormalities (43/57) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions. LOH hot-spots on chromosomes 3p, 4p and 6p found were confirmed by microsatellite analysis.ConclusionFrom our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Using flow cytometry and SNP-arrays, stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-011-0061-5) contains supplementary material, which is available to authorized users.

Updated clinical and biomarker results from a phase I study of vandetanib with radiation therapy (RT) with or without cisplatin in locally advanced head and neck squamous cell carcinoma (HNSCC).

5510 Background: Vandetanib (V) is an oral inhibitor of VEGFR, EGFR and RET signaling. Methods: Eligible patients (pts) with previously untreated, unresected HNSCC received once-daily V (starting dose 100 mg) for 14 days (d) followed by V + RT (2.2 Gy/d, 5 d/wk; total 66 Gy) for 6 wks (regimen 1) or V + RT (2 Gy/d, 5 d/wk; total 70 Gy) + cisplatin (30 mg/m2 weekly) for 7 wks (regimen 2). The primary objective was to determine the safety, tolerability and maximum tolerated dose (MTD) of V. We used multianalyte biomarker profiling (Luminex Corp) and ELISA to assess 57 plasma markers before and after 14 d of V and at completion of RT (d50), and EGFR, p16, E-cadherin and vimentin immunohistochemistry in tumor tissue. Clinical results were correlated with biomarkers. Results: Of 33 pts (median age 53 yrs; 28 male; all M0) who received treatment, 30 completed therapy (regimen 1, n=12; regimen 2, n=18). In regimen 1, there were no DLTs in the 100 mg cohort and 1 DLT in the 200 mg cohort (each n=6). In regimen 2, there were no DLTs in the 100 mg cohort and 3 DLTs in the 200 mg cohort (each n=6); 100 mg was defined as the MTD and the cohort expanded to 15 pts. The most common grade ≥3 AEs were dysphagia (27%), stomatitis (24%) and mucosal inflammation (21%); 4 pts discontinued V due to AEs. All 29 pts evaluable for efficacy (RECIST) achieved a complete response. At data cut-off, 11/29 had completed 2-yr follow-up without recurrence and 6 had recurrence. Steady-state exposure to V was unaffected by RT/cisplatin. Blood biomarkers were analyzed in 31 (baseline), 30 (d15) and 26 (d50) pts; 27 pts had tissue analyses. High levels of baseline plasma HGF, IL-8, MIF and eotaxin were associated with shorter progression-free survival (PFS; P<0.05). Significant increases in six plasma factors (HGF, OPN, IL-6, soluble IL-2R, IP-10 and MCP-1) were observed after RT, but not V alone. Tumor p16 expression was associated with superior PFS (P=0.01). Conclusions: The combination of V with RT ± cisplatin is tolerable with encouraging efficacy. Baseline HGF, IL-8, MIF and eotaxin are potentially useful markers of adverse outcome. Inflammatory and hypoxia-regulated cytokines appear to be upregulated after RT.

Disturbances in the positioning, proliferation and apoptosis of neural progenitors contribute to subcortical band heterotopia formation

Cortical malformations are commonly associated with intractable epilepsy and other developmental disorders. Our studies utilize the tish rat, a spontaneously occurring genetic model of subcortical band heterotopia (SBH) associated with epilepsy, to evaluate the developmental events underlying SBH formation in the neocortex. Our results demonstrate that Pax6+ and Tbr2+ progenitors are mislocalized in tish+/− and tish−/−- neocortex throughout neurogenesis. In addition, mislocalized tish−/− progenitors possess a longer cell cycle than wild type or normally-positioned tish−/− progenitors, owing to a lengthened G2+M+G1 time. This mislocalization is not associated with adherens junction breakdown or loss of radial glial polarity in the ventricular zone (VZ), as assessed by immunohistochemistry against phalloidin (to identify F-actin), aPKC-λ and Par3. However, vimentin immunohistochemistry indicates that the radial glial scaffold is disrupted in the region of the tish−/− heterotopia. Moreover, lineage tracing experiments using in utero electroporation in tish−/− neocortex demonstrate that mislocalized progenitors do not retain contact with the ventricular surface and that ventricular/subventricular zone (SVZ) progenitors produce neurons that migrate into both the heterotopia and cortical plate (CP). Taken together, these findings define a series of developmental errors contributing to SBH formation that differs fundamentally from a primary error in neuronal migration.

Experience with adrenal schwannoma in a Chinese population of six patients

Benign adrenal schwannoma is an extremely uncommon cause of incidentaloma. This article describes our experience with the diagnosis and treatment of adrenal schwannoma in a Chinese population of 6 patients. To our knowledge, this is the largest series of this uncommon adrenal tumor treated in a single center. From May 1999 to May 2009, 6 patients with incidentally discovered adrenal schwannoma were operated on at a tertiary referral hospital in Eastern China. Clinical details, radiographic features, laboratory examinations, pathological findings, and follow-up data of these patients were analyzed. Of the 6 patients, 4 were females (F) and 2 were males (M), with a mean age of 40.5 yr (range: 30-47, M:F=2:1). The 6 patients were managed with open unilateral adrenalectomy; 5 patients had schwannomas on the left side, and 1 on right side. Abnormal urine catecholamine was detected in 1 patient. The mean pathological size of these tumors was 4.3 ± 1.1 cm. The diagnosis of schwannoma was based on classic histological findings, and supported by immunohistochemistry of S-100, vimentin, and ABC positivity. In the follow- up of 47.5 ± 32.3 months, no recurrence and metastasis were observed. Although there may be some clues for radiological diagnosis of adrenal schwannoma, preoperative misdiagnosis is not infrequent. Those tumors may occasionally have endocrine function. Histological examination is the key of diagnosis, and surgical resection is the treatment of choice once malignancy cannot be excluded by preoperative analyses.

Prolonged osteogenesis from human mesenchymal stem cells implanted in immunodeficient mice by using coralline hydroxyapatite incorporating rhBMP2 microspheres

The local environment plays an important role in osteogenic tissue regeneration. Our previous studies have shown that xenogenic transplantation of human mesenchymal stem cells (hMSCs) alone into immunodeficient mice did not result in long-term bone formation. This study investigates whether bone formation can be prolonged by incorporating human mesenchymal stem cells in mineralized scaffolds together with controlled delivery of a growth factor, BMP2. A composite of coralline hydroxyapatite (CHA) with poly(lactic-co-glycolic acid) (PLGA)-encapsulated rhBMP2 was incorporated with hMSCs in vitro. After 2 weeks in vitro culture the constructs were implanted subcutaneously in CB17 scid beige mice and harvested 10 weeks after implantation. The mineralized tissues were stained by using a fluorescent marker, 5FAM-risedronate, followed by observation with fluorescence microscopy, histology, histomorphometry, mouse-anti-human vimentin immunohistochemistry, and scanning microscopy. The results showed that compared with control materials in which only fibrous tissue formed following implantation of coralline scaffolds, bone-like tissue formed within the CHA composite containing PLGA encapsulated rhBMP2 and hMSCs for up to 10 weeks after implantation. Human cells, identified by the human vimentin-specific monoclonal antibody were seen within the bone-like tissue. In conclusion, incorporation of hMSCs into CHA with controlled delivery of BMP showed prolonged bone formation in immunodeficient mice. Further research is required to optimize the growth factor delivery system and to understand the underlying cellular and molecular mechanisms involved.

Long-term cellular and regional specificity of the photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina

This study investigated the anatomical consequences of a photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina. Retinae were examined 2 weeks, 1, 3, and 6 months after systemic IAA injection. The retinae were processed using standard histological methods to assess the gross morphology and topographical distribution of damage, and by immunohistochemistry to examine specific cell populations in the retina. Degeneration was restricted to the photoreceptors and was most common in the ventral retina and visual streak. In damaged regions, the outer nuclear layer was reduced in thickness or eliminated entirely, with a concomitant loss of immunoreactivity for rhodopsin. However, the magnitude of the effect varied between animals with the same IAA dose and survival time, suggesting individual differences in the bioavailability of the toxin. In all eyes, the inner retina remained intact, as judged by the thickness of the inner nuclear layer, and by the pattern of immunoreactivity for protein kinase C-alpha (rod bipolar cells) and calbindin D-28 (horizontal cells). Müller cell stalks became immunoreactive for glial fibrillary acidic protein (GFAP) even in IAA-treated retinae that had no signs of cell loss, indicating a response of the retina to the toxin. However, no marked hypertrophy or proliferation of Müller cells was observed with either GFAP or vimentin immunohistochemistry. Thus the selective, long lasting damage to the photoreceptors produced by this toxin did not lead to a reorganization of the surviving cells, at least with survival as long as 6 months, in contrast to the remodeling of the inner retina that is observed in inherited retinal degenerations such as retinitis pigmentosa and retinal injuries such as retinal detachment.

Vimentin Reactivity in Renal Oncocytoma: Immunohistochemical Study of 234 Cases

The expression of vimentin in benign renal oncocytomas has been controversial. However, this is of clinical significance because immunostains may be used in differential diagnosis of renal tumors on limited biopsy specimens. Using different staining and analysis methods, we studied vimentin immunoreactivity in a large series of renal oncocytomas with a special emphasis on the immunoreactivity patterns. Immunohistochemical expression of vimentin has been used in the differential diagnosis of renal epithelial neoplasms. Although typically expressed in most renal cell carcinomas, the immunoreactivity of this intermediate filament in renal oncocytomas has been controversial. We studied vimentin immunoreactivity in a large series of 234 renal oncocytomas using 2 staining methods as well as manual and automated imaging analyses. We found that the focal vimentin immunoreactivity can be seen in most (72.6%) renal oncocytomas with vimentin-positive tumor cells usually found in the edge of a central scar or in small clusters scattered throughout the tumor. Computer-aided imaging analysis using ChromaVision Automatic Cellular Imaging System II confirmed the difference in vimentin immunoreactivity between oncocytoma and other renal neoplasms. Our study of vimentin immunohistochemistry in a series of renal oncocytomas, which to our knowledge is the largest ever published, showed focal vimentin positivity detected in most oncocytomas. Because the vimentin staining patterns in renal oncocytomas are different from those seen in clear cell or papillary renal cell carcinomas, we consider vimentin staining to be helpful in the differential diagnosis of oncocytoma from other renal tumor mimics. Furthermore, strong vimentin positivity in a renal cell neoplasm does not exclude the diagnosis of renal oncocytoma, particularly in a limited biopsy specimen.