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Unlabelled: The aim of this study was to investigate the late changes in the expression of tenascin-C (TN-C) in salivary glands (SG) following irradiation (IRR)., Materials and Methods: In 124 submandibular SG from 62 Wistar rats, the effect of IRR dose (fractionated IRR, 2 Gy per day, total dose of 20, 40, or 60 Gy), time since IRR (6 months vs. 12 months), and animal age (1 year vs. 1.5 years) on TN-C expression profile and its distribution pattern was investigated., Results: Expression of TN-C showed slight to moderate alterations in the irradiated specimens. The expression differed in frequency and degree among various tissue structures. The most striking finding was pronounced dose-dependent heterogeneity, with increases, decreases and fluctuations in staining., Conclusion: The staining of TN-C predominantly showed notable dose-dependent heterogeneity, persisting for up to 1 year after completion of IRR. Thus, these findings can be attributed to late radiation effects. The altered expression of tenascin-C may play at least a partial role in late radiogenic dysfunction of the submandibular SG.

Background: External irradiation (IRR) of advanced head and neck tumors often includes tissues of the larynx and trachea unaffected by cancer. In these normal tissues, both single-cell damage (necrosis, apoptosis, functional cell death) and interstitial damage (edema, fibrosis, vascular alterations, cellular infiltrations) resulting in tissue remodeling can occur, depending on various IRR parameters. However, reports on radiogenic intermediate filament protein alterations in laryngeal-tracheal tissues are very rare. In this study, we investigated the phenotypic characterization of the normal integrity-supporting cytokeratins (CK) and vimentin following a clinically relevant IRR protocol in laryngeal-tracheal tissues., Materials and Methods: In 61 laryngo-tracheal specimens from Wistar rats the expression profile and distribution pattern of CK (CK13, CK17/19, CK18) and vimentin were investigated according to IRR dose (fractionated IRR, 2 Gy per day, total dose of 20, 40 or 60 Gy), time from IRR (6 months vs. 12 months) and animal age (1 year vs. 1.5 years) using immunohistochemical methods, semiquantitative assessment and multivariate analysis., Results: In irradiated specimens, expression of both CK and vimentin showed slight to moderate dose-dependent alterations. The expression differed in frequency and level among the various tissue structures and showed remarkable heterogeneity, with increases, decreases and fluctuations in staining. In the glottic mucosal layer (non-keratinizing squamous epithelium), CK13 expression decreased with increasing dose. The CK17/19 expression of supra- and subglottic respiratory epithelia following 20 and 60 Gy exposure was significantly lower than in controls. The respiratory epithelia and, in part, the cuboidal epithelia of the indifferent type at the inner side of the aryepiglottic fold revealed increasing CK17/19 immunoreactions up to 40 Gy IRR, but a distinct decrease in expression at 60 Gy. In subglottic gland structures, CK18 was detected at significantly higher levels than in controls. There was increasing expression with increasing dose. CK18 reactions of supra- and subglottic respiratory mucosal layer, supraglottic gland structures and thyrocytes tended towards increasing expression with increasing dose and in older animals. Tracheal mucosal epithelia, tracheal glands, and respiratory epithelia of the inner side of the aryepiglottic fold tended towards decreasing expression of CK18 with increasing dose and in older animals. In part, these tissues showed dose-dependent fluctuations. Furthermore, the vimentin reactions showed dose-dependent, heterogeneous patterns, with increases, decreases, and fluctuations in staining. Moreover, there were differences in frequency and intensity of expression among the various tissue structures. Age and time from IRR had no significant effect on immunoreaction., Conclusion: The staining of CK and vimentin predominantly showed a notable dose-dependent heterogeneity, with increases, decreases and fluctuations in expression. The expression pattern persisted for up to 1 year after the completion of irradiation. Thus, these findings must reflect late radiation effects. The altered expression of CK and vimentin may play at least a partial role in structural (e.g. edema) and functional (e.g. voice disorders) changes associated with irradiation of the head and neck.

Objective: Basement membranes-(BM) influence polarization, differentiation, migration and proliferation of cell and play an important role in maintaining structural and functional tissue integrity. While BM alterations have been reported in various lesions (e.g. inflammation, tumors) of laryngeal-tracheal tissues, reports on radiogenic BM alterations are rare. External irradiation (IRR) of advanced head and neck tumors often includes "normal tissues" (tissues without cancer) of the larynx. In these normal tissues both single-cell damage (necrosis, apoptosis, functional cell death) and interstitial damage (edema, fibrosis, vascular alterations, cellular infiltrations) resulting in tissue remodeling can occur, depending on various IRR parameters. In this study, we set out to add to our knowledge on the phenotypic characterization of the radiogenic BM expression pattern in laryngo-tracheal tissues., Materials and Methods: In 63 laryngo-tracheal specimens from Wistar rats, we investigated the laminin (LA) and collagen IV (CIV) expression profile and distribution pattern depending on the IRR dose (fractionated IRR, 2 Gy/day, up to a total dose of 20, 40, or 60 Gy), the time since IRR (6 months vs 12 months) and animal age (1 year vs 1.5 years) using immunohistochemical methods, semiquantitative assessment, and multivariate analysis., Results: In specimens irradiated with more than 20 Gy, both BM constituents predominantly showed dose-dependent increases and sometimes fluctuations in staining at slight to moderate levels. The expression differed in frequency and level among the various tissue structures. In some structures there was decreased expression. In the vocalis muscle, laryngeal and esophageal nerve endings, recurrent laryngeal nerve and laryngeal and tracheal muscles, LA was detected at levels significantly stronger than in controls. BM surrounding gland structures, nerve endings of the piriform sinus and esophageal muscles showed a marked tendency towards increased LA expression. However, the BM underlying the mucosal layer of the supra- and subglottic region revealed decreasing LA immunoreaction up to 40 Gy IRR, but a distinct increase in expression at 60 Gy. In the esophageal and tracheal muscles, tracheal perichondrium, recurrent laryngeal nerve and capillaries, CIV was detected at significantly stronger levels than in the controls. The vocal ligament exhibited positive CIV immunoreactions adjacent to interstitial and infiltrate cells and CIV-positive BM condensations, resulting in increased staining of these structures. CIV reactions of laryngeal and hypopharyngeal nerve endings tended towards increased expression. In contrast, BM staining surrounding vocal muscle cells revealed significantly decreased expression. In addition, there was a tendency towards decreased expression for supraglottic, subglottic and hypopharyngeal muscle cells. Age and time since irradiation had no significant effect on staining., Conclusion: The BM constituents laminin and collagen IV showed prominent dose-dependent increases and sometimes fluctuations in expression. This expression pattern persisted up to one year after completion of the irradiation. Thus, these findings must be related to late radiation effects. The altered BM expression may play a role, at least in part, in structural (e.g. laryngeal edema) and functional (voice disorders) changes associated with irradiation of the head and neck area.

The role of the bcl-2 gene family members in promoting or antagonizing apoptosis in malignant tumors, including soft tissue sarcomas (STS), is well known. However, the impact of mRNA expression of bcl-2 family genes on prognosis has not been thoroughly investigated in STS. Samples from 82 STS patients were analyzed for mRNA expression of bad, bax, bcl-xL and bcl-2 by a high-throughput quantitative RT-PCR approach, using validated assays based on TaqMan technology. The mRNA data, related to glyceraldehyde-3-phosphate dehydrogenase expression measured in the same sample, were analysed for their correlation to tumor stage and overall survival of patients. In a Kaplan-Meier analysis none of the mRNA levels investigated differed significantly with regard to their impact on survival (log-rank test). However, after including the tumor stage in the statistical analysis, a borderline significance was observed for bad mRNA expression (p=0.068) indicating a stage-specific impact of mRNA expression on prognosis. Considering STS patients of tumor stage 2, multivariate Cox analysis revealed that bad mRNA values > or = 10 (p=0.0039; RR=9.08), bcl-xL > or = 1.5 (p=0.067; RR=4.59), bax > or = 0.005 (p=0.1; RR=2.84) and bcl-2 < 3 (p=0.42; RR=1.7) were associated with a poor prognosis. Combined high bad/bcl-xL mRNA expression levels revealed a 20-fold increase in the relative risk of tumor-related death (p=0.016) when comparing the poor and good prognosis groups. There was a 14.5-fold and 6.5-fold increase in the risk for the combinations of high bax/bcl-xL mRNA (p=0.018) and bax/bcl-2 mRNA expression (p=0.017), respectively. In conclusion, high bad mRNA levels and combined values of bad/bcl-xL bax/bcl-xL and bax/bcl-2 appear to be independent prognostic factors at least for stage 2 STS patients. In the combinations of mRNA levels there was more than an additive effect pointing to different pathways of prognostic relevance.

Objective: The extent of radiogenic salivary tissue damage (direct cell damage, reactive cell infiltration, increasing fibrosis) depends on the radiation dose (RD), the fractionation (FN) and the localisation of the salivary gland (SG) in the radiation field (RF). Because the histological damage is characterized by cellular and structural alterations and because extracellular matrix proteins, for example laminin (LA), are among the main components of the basement membrane (BM), which is considered to possess cell- and tissue structure-regulating functions, we set out to describe radiogenic BM changes., Materials and Methods: In 124 rat mandibular SG we investigated HE-stained sections and the LA staining profile (LASP) dependent on age [1 year (y) vs. 1 1/2 y], on FN [2 Gy/day up to a total dosage of 20/40/60 Gy (X-rays)], on RF [(left head and neck area); inside (left-sided SG) vs. outside (right-sided SG) RF] and on the latency of irradiation (1/2 y vs. 1 y) using immunohistochemical methods and multivariate analysis., Results: The immunoreactivity of the polyclonal antibody in the rat did not differ from the LASP in humans. LA was detected at variable levels in the glandular tissue, in vascular walls (without staining in the adventitia) and in nerve tissue (NT). No reaction was found in the capsule and the trabecula. In irradiated SG most tissue structures, including the vascular wall and NT, showed significantly stronger staining (both increased intensity and more extended expression). With increasing RD from 20 Gy to 60 Gy we found an increasing staining reaction. In addition, conglomerated LA depots were noted, especially in ares of disturbed or former acini of glands exposed to 60 Gy. The LASP up to 20 Gy was non-uniform and did not differ significantly from that in controls. A remarkable finding was the similar dose-dependent increase in staining reaction on both sides in several tissues of irradiated animals. Predominantly, however, we found considerably greater increases in staining in directly exposed glands than in out-of-field glands. Age and time since irradiation had no significant effect on staining., Conclusion: The BM constituent LA showed dose-dependent increases in expression--analogous to positively dose-dependent increasing SG tissue damage--which could contribute to disturbed cell-matrix interaction and SG radiation damage. Several parenchymal structures may be more sensitive to possible scattered radiation.

Objective: Laboratory rats can develop benign or malignant tumors (TM) spontaneously or following various carcinogenic processes, e.g. irradiation. The effects of irradiation vary according to the irradiation field (RF), the dosage and the strain of rat. Radiation-induced malignant TM in rats are predominantly sarcomas. Carcinomas, especially adenoid-cystic carcinomas (ACC) and adenocarcinomas of the head and neck region, are rarely reported in rats. The aim of this study was to add to the knowledge on ACC and adenocarcinomas in rats developing inside the RF and spontaneously. The TM arose in the course of studies on other questions of radiation effects following fractionated irradiation (2 Gy/day, 5 times a week up to a total dose of 60 Gy)., Methods: We investigated 22 TM (14 malignant, 8 benign) of 22 female Wistar rats. Ten malignant TM developed in the RF and 4 outside of the left head and neck area. The RF comprised the left neck, extending from left auricle to left clavicle and included the midline organs of the neck. Besides assessment of hematoxylin-eosin (HE)-stained sections, epithelial differentiation was investigated using cytokeratin (CK) antibodies against CK 5/6, CK 7, CK 8/18, CK 13/15/16, CK 17 and CK 20 and the LSAB-2 detection system., Results: Nine malignant TM originated from the major salivary glands (SG), a further three from the milk line and two from the maxilla. Using HE staining the pattern of rat malignant TM differed from that found in humans and was difficult to interpret. Two ACC, two cystadenocarcinomas, one microcystic adenocarcinoma and four squamous cell carcinomas (SCC) arising from the SG (one SCC was observed in the maxilla) developed in the RF. One microcystic adenocarcinoma, one ACC and one adenocarcinoma with sebaceous differentiation arising from the milk line and one SCC arising from the maxilla were found in non-irradiated animals. As typical results, in the ACC CK 17 was distinctly immunoreactive in excretory duct structures (ECD). CK 5/6 and CK 13/15/16 were marked at variable levels in myoepithelial cells (MC) and in basal cells of ECD. In the cystadenocarcinomas the ECD were clearly identified with CK 17 and CK 8/18 antibodies. MC and basal cells of ECD were positive for CK 5/6 and CK 13/15/16 antibodies., Conclusion: The CK expression profile of these rare and aggressive TM in rats differed according to the entity and SG structure. The differentiation markers were predominantly found in ECD and in modified MC. Concerning the growth pattern of the TM, the variation in size of the cysts and pseudocysts was remarkable. The unusual tumor features reduced the comparability with humans. The differentiation pattern did not differ noticeably between TM originating inside or outside the RF. Identification of CK subtypes in rat tumors facilitates their differential diagnosis.

Objective: The extent of radiogenic damage in salivary gland (SG) tissue depends on the radiation dose (RD), the fractionation (FN) and the localization of SG in the radiation field (RF). While the functional restriction and the radiogenic SG tissue damage are well documented using histomorphological, electron-microscopic and enzyme-histochemical methods, immunohistochemical analysis (IH) of cytokeratins (CK), epithelial differentiation markers, and vimentin, a marker of mesenchymal cells, are rare. Previous studies have shown stronger immunoreactivities of CK in irradiated glands exposed to 60 Gy total dosage. This study was performed to examine dose dependence and alterations related to age, RF, and latency of irradiation., Methods: In 124 rat mandibular SG we investigated the vimentin and CK staining profile dependent on age [1 year (y) vs. 1 1/2 y], on FN [2 Gy/day up to a total dosage of 20/40/60 Gy (x-rays)], on RF (inside vs. outside RF) and on the time since irradiation (1/2 y vs. 1 y) using IH., Results: The mouse monoclonal anti-CK antibodies [(AB) D5/16B4, Ks 13.1, E 3, K8.12, Ks 18.04, against CK 5-6, CK 13, CK 17, CK 13-15-16, CK 18) and the polyclonal anti-vimentin AB GP53 identified different epithelia and mesenchymal structures in rat SG tissue, including excretory duct cells (ECD), striated duct cells (SD), granular convoluted tubules (GTC), intercalated duct cells (ICD) and myoepithelial cells (MC). MC and mesenchymal cells were positive for vimentin AB. The different CK were detected in cell type-specific patterns and at variable levels in non-irradiated SG. In irradiated SG most cell types showed significantly stronger staining for various CKs. With increasing RD from 20 Gy to 60 Gy we found an increasing staining reaction. The CK staining profile up to 20 Gy was non-uniform and did not differ significantly from controls. Age and time since irradiation played a minor role or had no significant effect on staining., Conclusions: The CK and vimentin immunoreactivity showed dose-dependent increasing expressions, which could contribute to radiogenic cell and tissue damage. In some tissue structures a possible scattered irradiation effect should be mentioned. Age and time since irradiation (chosen in the study) had a minor or insignificant effect on staining profiles.

Objective: A variety of carcinomas (CA) can metastasize to the lymph nodes of the neck. Differentiation of CA in the lymph nodes according to their resemblance to the structure of origin is the basis of histopathological diagnosis. However, during the course of the disease, e.g. tumor recurrence after ablative surgery, these tissues can completely lose the ability to imitate typical structures of the organ that gave rise to malignant transformation. This can result in the inability of the pathologist to identify the origin of the metastases. The identification of the large group of cytokeratins (CK) as a member of the family of intermediate filaments has improved the diagnosis of epithelial tissues. An attempt was made to use CK antibodies to identify the organ of origin of poorly differentiated and anaplastic lymph node metastases of the neck., Methods: We investigated 34 routinely fixed (formalin/paraffin) lymph node metastases of the neck or specimens of these metastases. The tumors differed in terms of suspected primary tumor site and differentiation. Depending on the case history, diagnosis was performed by hematoxylin-eosin staining and by immunohistochemical staining of sections using antibodies against CK (CK nos. 1-10-11, 5-6, 6-8, 7, 8, 8-18, 13-15-16, 19 and 20) and against additional markers [vimentin, leukocyte common antigen (LCA), S-100, gross cystic disease fluid protein (GCDFP) and Epstein-Barr virus-induced latent membrane antigen (EBV-LMP)]., Results: The histopathological diagnosis was lympho-epithelial CA (8 cases), thyroid gland CA (2 cases), mammary gland CA (5 cases), bronchial CA (4 cases), basaloid CA (3 cases), clear cell CA (2 cases), sebaceous CA (1 case) and pharyngeal CA (9 cases). Some metastases were anaplastic in differentiation (G3-4). The marker expression in the immunohistological sections supported the histopathological findings. In some cases diagnosis succeeded especially in evaluating the marker expression. For example, in lympho-epithelial CA the epithelial tumor cell formations were positive for CK 5-6 antibodies, while expression in the lymphatic cells was lacking. In mammary gland CA the tumor cells were clearly identified by their positivity for CK 7 and GCDFP antibodies, in contrast to the negative infiltrating cells surrounding the tumor cells., Conclusions: In light of the case history details and the histopathological findings, immunohistological expression of various markers, especially the identification of CK subtypes, supported the differential diagnosis. In some cases the diagnosis may be established only by using CK profile and additional markers.

Objective: While radiation-induced sarcomas or carcinomas following chemical carcinogens are well-documented in rats, radiation-induced carcinomas, especially adenoid-cystic carcinomas (ACC) and adenocarcinomas, originating from the head and neck region, including the major salivary glands (SG) are rarely reported. Because in human ACC of the SG structural changes of the basement membrane (BM) with positive correlation of tumor differentiation loss and BM thinning have been described, this study set out to analyze collagen distribution in malignant rat tumors (TM) developing inside the radiation field (RF) and spontaneously. The TM arose in the course of studies on other questions regarding radiation effects following fractionated radiation (2 Gy/day, 5 times a week; total dose 60 Gy)., Methods: We investigated 22 TM (14 malignant, 8 benign) of 22 female Wistar rats. The RF comprised the left head and neck area. Besides assessment of hematoxylin-eosin (HE)-stained sections collagens (C; types III and IV) were investigated using immunohistochemical methods., Results: Nine malignant TM originated from the SG, a further three from the milk line and two from the maxilla. Two ACC, two cystadenocarcinomas, one microcystic adenocarcinoma and four squamous cell carcinomas (SCC) arising from the SG (one SCC was observed in the maxilla) developed in the RF. One microcystic adenocarcinoma, one ACC and one adenocarcinoma with sebaceous differentiation arising from the milk line and one SCC arising from the maxilla were found in non-irradiated animals. As typical results, in the ACC (glandular subtype), C III was detected in the interstitium with sometimes stronger staining surrounding myoepithelial cells (MC) and excretory duct structures (ECD). Weak C IV staining in a string-like fashion was found in ECD and MC. In larger pseudocysts the lumen contained substances reacting with C IV antibodies. In the cystadenocarcinomas and the microcystic adenocarcinomas reactions at variable levels after anti-C III incubation were found close to modified MC and ECD with transition to the interstitium. C IV was more intensely stained in these entities, in part continuously and with broadening around MC and ECD. However, especially in more anaplastic parts of the tumor, fragments, interruptions or loss of the BM were noted. Focal interstitial immunoreactivity, e.g. conglomerates, was also identified., Conclusion: In rat carcinomas collagen detection was partially of a continuous layer, even with BM thickening and more extended deposition. In contrast, BM fragmentation or loss was displayed more often in anaplastic parts of the tumor. Also, the interstitium showed conglomerated collagen formations. Therefore, the increasing loss of BM in rat SG tumors is similar to that in humans and in both species is a sign of dedifferentiation.

Background: The aim of this study was to compare sequencing and immunohistochemistry (IHC) for their ability to detect p53 mutations and to assess their prognostic value in soft tissue sarcomas (STS)., Material and Methods: 146 STS samples were investigated by single strand DNA conformation polymorphism (SSCP)-sequencing for p53 mutations. Additionally, IHC with five p53 antibodies (CM-1, Pab1801, Pab240, DO1, DO-7) was performed., Results: In 65 to 131 tumor samples (44.5% to 90.4%) elevated levels of p53 protein were recorded by IHC, depending on the antibody applied. Sixteen out of 146 STS tumor samples (11%) had p53 gene mutations. 13 cases (81.2%) of the 16 tumors with p53 mutations could be detected by DO-1. However, of the 93 DO-1 positive tumors 79 were negative by SSCP-sequencing analysis. Using multivariate regression analysis (Cox proportional hazards model) both p53 mutations (non-frameshift mutations) and IHC detection of p53 protein overexpression were prognostic predictors for poor survival., Conclusions: This study suggests that IHC is valuable for assessing p53 mutations in STS, but sequencing provides additional important information on the molecular characteristics of the alterations.

Chemical conjugation of carbohydrate derivatives to a labelled carrier protein makes it possible to monitor the presence of specific sugar-dependent binding to routinely processed tissue sections. Different levels of cytoplasmic staining were observed in normal urothelium for the applied probes that expose a beta-D-galactoside, alpha- or beta-N-acetyl-D-galactosaminide, beta-N-acetyl-D-glucosaminide, alpha-D-mannoside, alpha-L-fucoside or a-D-glucoside, respectively. These moieties are crucial parts of the recognition sites in cellular glycoconjugates for plant lectins. The extent of staining invariably increased from the basal to the luminal cell layer. No nuclear staining was observed. Within the 18 cases of bladder lesions studied, the appearance of nuclear staining was characteristic of transitional cell carcinoma, its correlation to the histologic grade being dependent on the type of neoglycoprotein. Cytoplasmic staining revealed at least two patterns of grade-dependent variations, namely rather constant expression for lactose-mannose-, fucose- and maltose-specific sites and an initial increased followed by a loss of binding activity at the highest level of cellular atypia for the other three types of probe. Intratumoral heterogeneity was especially visible in tumors of this group. Interestingly, the known positive correlation of presence of the Thomsen-Frieden rich antigen with the level of cellular atypia, monitored with peanut agglutinin, could be extended to the expression of respective receptor sites, which was experimentally feasible due to the synthesis of this disaccharide structure and its immobilization to the carrier protein.