Identification of SEC62 as a potential marker for 3q amplification and cellular migration in dysplastic cervical lesions.

BACKGROUND: Chromosome 3 amplification affecting the 3q26 region is a common genomic alteration in cervical cancer, typically marking the transition of precancerous intraepithelial lesions to an invasive phenotype. Though potential 3q encoded target genes of this amplification have been identified, a functional correlation of potential oncogenic function is still missing. In this study, we investigated copy number changes and the expression level of SEC62 encoded at 3q26.2 as a new potential 3q oncogene in dysplastic cervical lesions and analyzed its role in cervical cancer cell biology. METHODS: Expression levels of Sec62 and vimentin were analyzed in liquid based cytology specimens from 107 women with varying grades of cervical dysplasia ranging from normal cases to cancer by immunofluorescence cytology. Additionally, a subset of 20 representative cases was used for FISH analyses targeting SEC62. To further explore the functional role of Sec62 in cervical cancer, HeLa cells were transfected with a SEC62 plasmid or SEC62 siRNA and analyzed for their proliferation and migration potential using real-time monitoring and trans-well systems as well as changes in the expression of EMT markers. RESULTS: FISH analyses of the swabbed cells showed a rising number of SEC62 gains and amplifications correlating to the grade of dysplasia with the highest incidence in high grade squamous intraepithelial lesions and squamous cell carcinomas. When analyzing the expression level of Sec62 and vimentin, we found a gradually increasing expression level of both proteins according to the severity of the dysplasia. In functional analyses, SEC62 silencing inhibited and SEC62 overexpression stimulated the migration of HeLa cells with only marginal effects on cell proliferation, the expression level of EMT markers and the cytoskeleton structure. CONCLUSIONS: Our study suggests SEC62 as a target gene of 3q26 amplification and a stimulator of cellular migration in dysplastic cervical lesions. Hence, SEC62 could serve as a potential marker for 3q amplification, providing useful information about the dignity and biology of dysplastic cervical lesions.

Noninvasive histological imaging of head and neck squamous cell carcinomas using confocal laser endomicroscopy.

Confocal laser endomicroscopy (CLE) is an imaging technique that uses miniaturized fiberoptic probes to allow real-time histological imaging of human tissue. An application of CLE in otorhinolaryngology has hardly been investigated so far. In our study, we analyzed the applicability of CLE to visualize cancerous and healthy tissue of the head and neck region. Formalin-fixed tissue specimens from 135 head and neck squamous cell carcinoma (HNSCC) patients and 50 healthy controls were investigated using CLE with and without topical application of acriflavine. Four head and neck surgeons, four pathologists, and four laymen evaluated the CLE images of the HNSCC cases regarding the tumor localization and its border to healthy tissue. The tumor localization and the tumor border were correctly identified in 97 % by the pathologists, 85 % by the head and neck surgeons, and 70 % by the laymen. The main difference in evaluation results was seen in the correct identification of the tumor site (p < 0.05), while there was no significant difference in the identification of the tumor border. CLE is a valuable tool for real-time histological imaging of HNSCCs. It can help to visualize the tumor border and, thereby, facilitate a more precise tumor surgery.

Orthotopic tumorgrafts in nude mice: A new method to study human prostate cancer.

BACKGROUND: In vivo model systems in prostate cancer research that authentically reproduce tumor growth are still sparse. While orthotopic implantation is technically difficult, particularly in the mouse, most models favor subcutaneous tumor growth. This however provides little information about natural tumor growth behavior and tumor stroma interaction. Furthermore, established prostate cancer cell lines grown as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behavior in men. Primary cell cultures are difficult to handle and an induction of orthotopic tumors has not been successful yet. Therefore, a tumorgraft model using tumor tissue from prostatectomy specimens was developed. METHODS: Balb/c nude mice were used to graft fresh prostate tumor tissue by renal subcapsular and orthotopic implantation. Testosterone propionate was supplemented. Animals were tracked by means of 30 MHz ultrasound to monitor tumor engraftment and growth. Autopsy, histology, PSA measurements as well as immunostaining and PCR for human tissue were performed to confirm orthotopic tumor growth. RESULTS: Renal subcapsular engraftment was seen in 2 of 3 mice. Orthotopic engraftment was observed in 7 of 11 animals (63.6%) with an overall engraftment of 5 out of 9 patient specimens (55.6%). Ultrasound confirmed the tumor growth over time. Of interest, the tumorgrafts not only retained essential features of the parental tumors, but also stained positive for tumor specific markers such as AR, PSA, and AMACR. Tumor positive animals showed highly elevated serum PSA levels with confirmation of a human specific PCR sequence and a human endothelial cell lining in the tumor vessels. CONCLUSIONS: Standardized implantation of fresh tumor tissue in nude mice prostates generates tumorgrafts with histological properties of organ-confined prostate cancer. These tumorgrafts display a new approach for an optimized in vivo model of prostate cancer and will allow further investigations on specific pathways of tumor initiation and progression as well as therapeutic response.

Long-Term Follow-Up of Pediatric Excimer Laser-Assisted Penetrating Keratoplasty for Congenital Stromal Corneal Dystrophy.

PURPOSE: The purpose of this study was to highlight characteristic clinical and microscopic findings and report the long-term follow-up of pediatric excimer laser-assisted penetrating keratoplasty (excimer-PKP) for congenital stromal corneal dystrophy (CSCD). METHODS: A 2-year-old Greek child presented with CSCD at our department. Clinical examination showed bilateral flake-like whitish corneal opacities affecting the entire corneal stroma up to the limbus. Genetic testing identified a mutation of the decorin gene (c.962delA). The variant was not present in the parents and represented a de novo mutation. The uncorrected visual acuity was 20/100 in both eyes. Excimer-PKP (8.0/8.1 mm) was performed on the right eye at the age of 2.5 years and on the left eye at the age of 3 years. Postoperatively, alternating occlusion treatment was performed. RESULTS: The light microscopic examination demonstrated a disorganized extracellular matrix of the corneal stroma characterized by a prominent irregular arrangement of stromal collagen lamellae with large interlamellar clefts containing ground substance, highlighted by periodic acid-Schiff- and Alcian blue-positive reaction detecting acid mucopolysaccharides. Electron microscopy showed disorganization and caliber variation of collagen lamellae and thin filaments within an electron-lucent ground substance. The postoperative course was unremarkable. Both grafts remained completely clear 14 years postoperatively. Corneal tomography showed moderate regular astigmatism with normal corneal thickness. The corrected distance visual acuity was 20/25 in both eyes. CONCLUSIONS: Excimer-PKP for CSCD might be associated with excellent long-term results and a good prognosis, particularly when the primary surgery is performed at a very young age. However, this requires close postoperative follow-up examinations by an experienced pediatric ophthalmologist to avoid severe amblyopia.

Progranulin antibodies in autoimmune diseases.

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), Behcet's disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

Changing indications for penetrating keratoplasty in Homburg/Saar from 2001 to 2010--histopathology of 1,200 corneal buttons.

BACKGROUND: To evaluate the trends in indications for penetrating keratoplasty (PKP) in Homburg/Saar between 2001 and 2010. METHODS: Retrospective review of 1,200 corneal buttons which underwent PKP that were performed between 2001 and 2010 at the Department of Ophthalmology of Saarland University Hospital, Germany. Indications were classified into eight different groups following histological analysis: keratoconus, Fuchs' dystrophy, bullous keratopathy, corneal scars, keratitis, regraft, corneal dystrophy other than Fuchs' dystrophy, and other diagnoses. Two different time periods (between 2001-2005 and between 2006-2010) were analyzed. RESULTS: Keratoconus (25.5 %) was the most common indication for PKP in our study, followed by Fuchs' dystrophy (21.2 %), bullous keratopathy (14.6 %), corneal scars (14.4 %), keratitis (13.0 %), regraft (7.0 %), non-Fuchs' dystrophies (2.1 %), and other diagnoses (2.3 %). Comparing the two different time periods, a trend of significantly increasing frequency of keratoconus and Fuchs' dystrophy, and a decreasing frequency of corneal scars, were found as indications for PKP in our study. CONCLUSIONS: Keratoconus was the leading indication for PKP in our series, and had a significantly increasing trend from 2001-2005 to 2006-2010. The percentage of patients with Fuchs' dystrophy increased, and became the second most common indication for PKP, while the number of PKPs for corneal scars decreased during the last 5 years in our institution.

Histological and biochemical findings in membranous cataract.

BACKGROUND: In this report we present a patient with unilateral membranous cataract and describe the histological and biochemical findings accompanying this rare condition. METHODS: The patient underwent an uneventful cataract extraction. Aqueous humor (20 µl) was aspirated from the anterior chamber intraoperatively and processed for fibroblast growth factor (FGF) and epidermal growth factor (EGF) using an immunoassay method (ELISA). The lens material was subjected to histological examination. RESULTS: The patient had increased levels of FGF and EGF in the aqueous humor, as measured by ELISA. Histological examination of the lens material showed a marked fibrous metaplasia and thickening of the anterior lens capsule, while the lens epithelial cells were transformed to active myofibroblasts which generated a fibrous matrix of collagen lamellae. Unfortunately, visual function was not restored postoperatively due to underlying amblyopia. CONCLUSIONS: Our histological and biochemical findings suggest that FGF and EGF may play a key role in the formation of membranous cataract, and therefore their impact on lens physiology should be further investigated.

Silencing of the SEC62 gene inhibits migratory and invasive potential of various tumor cells.

Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post-translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over-expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor-free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de-differentiation of the cells. Therefore, up-regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi-tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor-related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell-line PC3 as well as in a set of other tumor cell-lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.

Histology and function: analyzing the uvular muscle.

OBJECTIVE: Virtual surgery and virtual patients necessitate quantitative data on the area of interest. The study was conducted to exactly describe the embryonic and fetal uvular muscle (MU), relevant for clinical as well as virtual surgery and virtual patient generation. METHOD: Serially sectioned viscerocrania of 10 aborted embryos and fetuses underwent three-dimensional reconstruction to obtain detailed anatomic data and perform finite element analyses. RESULTS: The MU was paired in 80% of cases, while 20% allowed no clear-cut distinction. The MU merged with the levator muscle beneath the palatal aponeurosis without a hard palate insertion. Superior longitudinal central fibers ran below the nasal mucosa, and few circular peripheral fibers crossed in the central third to the contralateral side. This was seen in 30% of the paired muscles and in all cases when no differentiation was possible; about 40% to 80% MU fibers crossed to the ipsilateral and contralateral palatopharyngeus muscle behind the levator loop. MU fibers inserted 60% nasal and 40% oral to the basal membrane at the middle third of the macroscopic uvula, made of loose connective tissue and salivary glands. The results of the finite element simulation of the uvula showed no distinct patterns or distributions of local stress. CONCLUSIONS: Detailed anatomical study supported the concept of mediocranial MU repositioning during corrective surgery, although the impact is minor to the levator muscle's action. Future mathematical models describing effects of such a maneuver should integrate surrounding structures.

Recurrence of macular corneal dystrophy on the graft 50 years after penetrating keratoplasty.

Purpose: To report the recurrence of a macular corneal stromal dystrophy 50 years after penetrating keratoplasty (PKP). Methods: Observational case report Case description: A 76-year-old male patient presented with visual impairment in the right eye (OD) 50 years after PKP in 1962 (44 years after PKP also in the left eye (OS) in 1968) following explosion injury. His visual acuity had already been impaired before the trauma because of bilateral corneal opacities. The central corneal thickness of the graft measured 584 µm (OD) and 544 µm (OS), whilst the peripheral host thickness (8 mm zone), however, was 1233 µm (OD, cranial) and 1131 µm (OS, nasal). The original graft diameter measured 6 mm in both eyes and the recipient cornea was cloudy and gray. The endothelial cell count was measured centrally (OD 1162 c/mm2, OS 1320 c/mm2). The visual acuity was 20/100 (OD) and 20/40 (OS). After excimerlaser-assisted repeated PKP (8.0/8.1 mm, OD), the histological analysis of the former graft revealed deposits of acid mucopolysaccharides (AMP) subepithelially, within the interface, in the donor stroma, and in the endothelium, which proved the peripheral recurrence of a macular corneal stromal dystrophy on the graft. Conclusion: Recurrence of macular corneal stromal dystrophy is seldom, but it may occur many decades after PKP. In this patient, the host's stroma was twice as thick as that of the graft. This may be caused by the active production of acid mucopolysaccharides in the host endothelium with secondary endothelial decompensation. Thus, PKP remains the gold standard in the cure of macular corneal dystrophy for long-term visual rehabilitation.

Effect of 3q oncogenes SEC62 and SOX2 on lymphatic metastasis and clinical outcome of head and neck squamous cell carcinomas.

Chromosome 3q26 amplification represents a frequent alteration in head and neck squamous cell carcinomas (HNSCCs). Overexpression of 3q26 encoded genes SEC62 and SOX2 was detected in various cancers, including HNSCCs, indicating their potential function as oncogenes. In our study, we elucidated the function of SEC62 and SOX2 in HNSCC patients, with a main focus on their effect on lymphatic metastasis and patient survival. We analyzed SEC62 and SOX2 expression in tissue specimens from 65 HNSCC patients and 29 patients with cervical cancer of unknown primary (CUP); a higher SEC62 and lower SOX2 expression was observed in the lymph node metastases from HNSCC patients compared with the respective primary tumor. Lymph node metastases from CUP patients showed higher SEC62 and lower SOX2 expression compared with lymph node metastases from HNSCC patients. When proceeding from the N1 to the N3 stage, SEC62 expression in the lymph node metastases showed an increase and SOX2 expression showed a decrease. Moreover, both genes showed a highly significant relevance as prognostic biomarkers, with the worst prognosis for patients with high SEC62 and low SOX2 expression levels. In functional analyses, knockdown of SEC62 resulted in an inhibition of HNSCC cell migration while, conversely, SEC62 and SOX2 overexpression stimulated cell migration. Taken together, our study showed that the expression of the 3q oncogenes SEC62 and SOX2 affects lymphatic metastasis and cell migration in HNSCC and CUP patients and has a high prognostic relevance in these diseases.

Characterization of miR-146a and miR-155 in blood, tissue and cell lines of head and neck squamous cell carcinoma patients and their impact on cell proliferation and migration.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies worldwide with an unchanged 5-year survival rate during the last decade. To detect reliable prognostic markers and improve patients' outcome in future, the aim of our study was to detect differences in microRNA (miRNA; miR) expression profile and further on to analyze the functional role of selected miRNAs. METHODS: Blood samples from HNSCC patients and sex- and age-matched healthy volunteers were analyzed by microarrays and validated by quantitative real-time PCR. Data were compared with tumor tissue results and all findings were correlated with clinical parameters. Additionally, the proliferation and migration potential of two cell lines transfected with miRNA mimics and inhibitors for miR-146a and miR-155 were examined. RESULTS: Initial analysis of blood samples showed no significant differences between the miRNA profile of HNSCC patients and healthy controls (p > 0.05). Interestingly, down-regulation of miR-146a and miR-155 in blood of patients correlated with the occurrence of distant metastasis regarding tumor patients only (p = 0.023 and p = 0.028, respectively). Additionally, our investigations in tissue samples revealed a lower expression of miR-155 in tumor cells (p = 0.003) and a correlation with higher cT-classification for down-regulation of miR-146a (p = 0.005). Moreover, functional assays demonstrated that inhibition of miR-146a and miR-155 promoted dramatically proliferation and migration potential, whereas transfection of both mimics had an inhibitory effect. CONCLUSIONS: Characterizing the expression of miR-146a and miR-155 and their functional role in tumor biology underlined significantly their proliferation and migration potential suggesting relevance as potential prognostic markers in HNSCC.

Differential expression of IGF components and insulin receptor isoforms in human seminoma versus normal testicular tissue.

Insulin-like growth factors (IGF) have mitogenic and antiapoptotic functions, and may be involved in tumor growth. The purpose of the study was to investigate the role of IGF components in seminoma compared to normal testis. Normal testicular tissues from autopsy cases and seminoma from surgery cases were obtained for microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of IGF-1, IGF-2, IGF receptor type 1 (IGF-R1), IGF-R2, insulin receptor isoforms A (IR-A) and B (IR-B), and IGF-binding proteins (IGFBP) 1-6. IGF-2 was localized by immunohistochemistry. IGFBP-5 protein expression was evaluated by Western blot analysis. mRNA expression in microarray and real-time RT-PCR showed similar tendencies: IGF-1, IGF-R1, IGF-R2, IR-A, and IGFBP-2 were not different in both groups. IGF-2, IR-B, IGFBP-1, IGFBP-4, and IGFBP-6 mRNA were downregulated in seminoma. IGFBP-3 tended to be upregulated in pT1 seminoma, but downregulated in pT2 stages. IGFBP-5 and IGF-2 protein expression correlated with mRNA expression. In conclusion, downregulation of mainly inhibiting IGFBPs may allow a stimulated tumor growth. The downregulated IGF-2 does not seem to be involved in the growth regulation of seminoma. Constantly expressed genes (e.g., IGF-1, IGF-R1, IR-A, and IGFBP-2) may reflect an involvement in spermatogenesis, but may also play a major role in tumor growth as their expression is not downregulated despite the lack of spermatogenesis in tumor tissue.

Combination of p16(INK4a) /Ki67 immunocytology and HPV polymerase chain reaction for the noninvasive analysis of HPV involvement in head and neck cancer.

BACKGROUND: High-risk human papillomavirus (HPV) infection has been identified as a relevant risk for the development of head and neck squamous cell carcinomas (HNSCCs). As HPV status has also gained a role as a prognostic and predictive biomarker for this entity, there is a growing demand for valid HPV testing in HNSCC patients METHODS: Liquid-based cytological smears from 45 HNSCC and 20 control patients were collected and used for simultaneous immunocytochemical p16(INK4a) /Ki67 staining using a CINtec PLUS kit after the presence of tumor cells was verified in a Papanicolaou-stained slide. The same cytological suspension was used for the detection of HPV DNA by specific polymerase chain reaction (PCR). RESULTS: Tumor cells were detected in the swab material of 44 HNSCC patients corresponding to a sensitivity of 98% (44 of 45). PCR analysis revealed the presence of HPV DNA in the cytological suspension of 13 patients (13 of 65, 20%) with simultaneous p16(INK4a) /Ki67 expression by the tumor cells in 11 of these HPV DNA-positive samples (11 of 13, 85%) - a staining pattern that is strongly associated with a carcinogenic HPV infection. CONCLUSIONS: A simultaneous immunocytochemical detection of p16(INK4a) and Ki67 can reliably be performed on liquid-based cytological smears from HNSCC patients using a CINtec PLUS kit. In addition, the same cytological material can be used for the detection of HPV DNA by specific PCR. The combined results of both techniques enable better discrimination between latent and carcinogenic HPV infections as well as HPV-negative cases and thus can provide information on the prognosis of HNSCC patients and facilitate therapeutic decisions.

The Fuhrman grading system has no prognostic value in patients with nonsarcomatoid chromophobe renal cell carcinoma.

The prognostic value of the Fuhrman nuclear grading system has been questioned for chromophobe renal cell carcinoma (chRCC) because this subtype frequently displays nuclear and nucleolar pleomorphism. The present study reevaluates this grading system in a series of patients with nonsarcomatoid chRCC. We identified 176 patients (3.6%) with nonsarcomatoid chRCC in a total of 4897 patients who underwent surgery for renal cell carcinoma at 5 centers in Germany between 1990 and 2010. The mean follow-up was 51.1 months. The 3 groups (G1 versus G2 versus G3/4) were comparable in terms of age, sex, tumor diameter, and lymph node metastasis. They only differed significantly in tumor stage (P = .01) and the incidence of synchronous visceral metastasis (P = .04). The 5-year cancer-specific survival rates were 84.4% for G1 (n = 32), 84.3% for G2 (n = 108), and 74.1% for G3/4 tumors (n = 33) (P = .58). Accordingly, multivariate analysis including age, sex, tumor stage, and metastatic disease did not identify Fuhrman grading as an independent predictor of cancer-specific survival in patients with chRCC (P = .4). We were able to demonstrate in a large multicenter cohort that the Fuhrman grading system does not qualify as a prognostic tool in patients with chRCC.

Repeat corneal graft failure due to graft-to-host herpetic infection.

BACKGROUND: Herein, we present the case of a young female patient with keratoconus, who was subjected twice to repeat keratoplasty, and each time, she experienced a corneal graft failure. FINDINGS: Under the suspicion of herpetic eye disease, we administered topical and systemic anti-herpetic treatment after the second repeat keratoplasty. The postoperative course was uneventful, and the corneal graft is clear, until recently. Immunohistochemistry and DNA-polymerase chain reaction were negative for herpes simplex virus-1 (HSV-1) in the host cornea, but they detected HSV-1 in both transplanted corneal grafts, thereby supporting our clinical hypothesis that graft-to-host HSV-1 infection elicited this chain reaction of complications in our patient. CONCLUSION: This clinical report illustrates in a unique way the dramatic impact an unsuspected herpetic infection in the corneal graft in cases of keratoplasty may have and underscores the necessity of suspecting and adequately treating these distinct cases.