Gastrointestinal adverse reaction to food (GARF) and endoscopic confocal laser endomicroscopy (eCLE).

BACKGROUND AND STUDY AIMS: Gastrointestinal adverse reaction to food (GARF) is reported frequently in the general population and even more in patients with disorders of the gut brain axis. However, there is a significant difference between self-reported and objective proven GARF. The aim of the study was to characterize a mucosal correlate of GARF by endoscopic confocal laser endomicroscopy (eCLE) with duodenal food challenge (DFC). PATIENTS AND METHODS: In an observational and proof of concept study we evaluated 71 patients with disorders of the gut brain axis without (group I, n=19) and with (group II, n=52) GARF by eCLE and DFC. Spontaneous and food induced transfer of fluorescein into duodenal lumen was detected 10 minutes following intravenously application of fluorescein and 10 minutes after DFC. RESULTS: According to Rom IV, the patients (group I/II) could be classified as irritable bowel syndrome (IBS) 32%/31%, functional abdominal pain without changes in bowel movement 47 %/48 %, functional abdominal bloating/distension 0 %/10 %, functional diarrhea 5 %/ 2 %, and unspecified functional bowel disorder 16 %/10 %, respectively. 21 %/27 % of the patients responded with a fluorescein leakage into the duodenal lumen before and 74 %/69 % following to DFC. Frequency rank order of food components that induced a response were soy (55.5 %/60 %), wheat (60 %/45.5 %), egg (35.7 %/8.3), milk (30 %/18.2 %) and yeast (10 %/6.6 %), respectively. Histology of duodenal biopsies, number, form and distribution of intraepithelial lymphocytes and mucosal mast cells as well as mast cell function were normal. Overall, 14 %/79 % reported main symptom benefit following a food exclusion therapy according to eCLE and DFC that was significant different between the groups. CONCLUSION: The results of our study indicate that eCLE with DFC is a technique to clinically evaluate patients with disorders of the gut brain axis and GARF resulting in a high proportion of patients reporting symptom benefit upon food exclusion dietary advice focussed on the results of eCLE.

Eosinophilic esophagitis and duodenal food challenge - evaluation through endoscopic confocal laser endomicroscopy.

BACKGROUND: Endoscopic confocal laser endomicroscopy (eCLE) is an established technique that allows clinical evaluation of mucosal integrity by fluorescein leaking through the mucosa upon duodenal food challenge (DFC). Analysis of eCLE with DFC in eosinophilic esophagitis (EoE) would be interesting to evaluate epithelial barrier dysfunction also in other regions of the gastrointestinal tract and to characterize potential individual food allergens that trigger the esophageal inflammation. METHODS: In an observational and proof of concept study we evaluated 9 patients with histologically proven EoE by eCLE and DFC. Severity of symptoms were graduated according to the validated symptom-based EoE activity index. The endoscopic appearance of the esophagus was described according to the Endoscopic Reference Score System (ERERS). Spontaneous and food induced transfer of fluorescein into duodenal lumen were detected 10 minutes following intravenously application of fluorescein and 10 minutes after DFC. Food allergens were yeast, egg, soy, milk, and wheat, respectively. Local application of sodium chloride solution 10 % to the duodenal mucosa before DFC served as a control. Patients responding to DFC received a dietary exclusion therapy according to the results of DFC. RESULTS: We investigated 9 patients with EoE (8 men, 49.7±13.8, 36-76 years). Symptom-based EoE activity index was 79±27.4, 33-100. In all patients EoE was confirmed by histology with number of esophageal mucosal eosinophilic granulocytes > 15/HPF, (91.4±77.4, 42-263). Mean ERERS score was 4.5±1.3, 3-7. None of the patients was aware of any food intolerance. eCLE revealed one patient with spontaneous transfer of i. v. fluorescein into duodenal lumen before DFC ("leaky gut"). 40 DFC were performed in the remaining 8 patients of whom 5 patients (61 %) responded to DFC. Rank order of fluorescein leakage upon DFC was wheat and milk in 37.5 % each, soy in 25 %, and egg in 12.5 %. The patients were treated by PPI (n=9), esophageal bouginage (n=5) and/or local corticoid therapy (n=3). The 5 patients responding to DFC received an additional food exclusion dietary advice focussed on the results of DFC. All patients reported a reduction of their symptoms. EoE activity indexes of patients with positive DFC were 73.7+28.6, 33-100 before and 22.7+37.9, 0-79 four weeks after food exclusion. CONCLUSION: The findings of our proof of concept study suggest that eCLE with DFC may be an interesting tool to further evaluate patients with EoE. This technique has the potential to identify patients who may benefit from an additional individual dietary therapy.

Is CCL2 an Important Mediator of Mast Cell-Tumor Cell Interactions in Oral Squamous Cell Carcinoma?

In this study, we aimed to evaluate the influence of interactions between mast cells (MCs) and oral squamous cell carcinoma (OSCC) tumor cells on tumor proliferation and invasion rates and identify soluble factors mediating this crosstalk. To this end, MC/OSCC interactions were characterized using the human MC cell line LUVA and the human OSCC cell line PCI-13. The influence of an MC-conditioned (MCM) medium and MC/OSCC co-cultures on the proliferative and invasive properties of the tumor cells was investigated, and the most interesting soluble factors were identified by multiplex ELISA analysis. LUVA/PCI-13 co-cultures increased tumor cell proliferation significantly (p = 0.0164). MCM reduced PCI-13 cell invasion significantly (p = 0.0010). CC chemokine ligand 2 (CCL2) secretion could be detected in PCI-13 monocultures and be significantly (p = 0.0161) increased by LUVA/PCI-13 co-cultures. In summary, the MC/OSCC interaction influences tumor cell characteristics, and CCL2 could be identified as a possible mediator.

Food intolerance in patients with functional abdominal pain: Evaluation through endoscopic confocal laser endomicroscopy.

Background and study aims Gastrointestinal symptoms assumed to be caused by food intolerance are reported frequently in the general population. There is a significant difference between self-reported and objective proven food intolerance, as shown by placebo-controlled, double-blind, randomized trials. This discrepancy may be overcome by endoscopic confocal laser endomicroscopy (eCLE). Patients and methods In an observational study we evaluated 34 patients with functional abdominal pain and adverse reaction to food by eCLE and local duodenal food challenge for the first time. Spontaneous and food-induced transfer of fluorescein into the duodenal lumen was detected 10 minutes after intravenously application of fluorescein and 10 minutes after duodenal food challenge (DFC). Results Of the patients, 67.6 % responded with a fluorescein leakage into the duodenal lumen. Frequency rank order of food antigens that induced a response were soy (50 %), wheat (46.1 %), milk (20 %), egg (12 %), and yeast (11.5 %), respectively. Of the patients, 23.5 % showed spontaneous leakage of fluorescein, suggesting leaky gut syndrome. Histology of duodenal biopsies and mast cell function were normal. Overall, 69.5 % of patients improved with food exclusion therapy and 13 % were symptom-free according to eCLE. Conclusions The results of our study indicate that eCLE is a clinically useful tool to evaluate patients with functional abdominal pain and adverse reaction to food and to create individualized dietary therapy with clinical benefit for patients.

Endoscopic laser endomicroscopy and "leaky gut" in patients with functional gastrointestinal symptoms and food intolerance.

BACKGROUND: Intestinal epithelial barrier dysfunction ("leaky gut syndrome", LGS) is thought to play a major role in the pathogenesis of disorders of the gut brain axis. Endoscopic confocal laser endomicroscopy (eCLE) is an objective measure to test duodenal permeability. We applied this technique in patients with functional gastrointestinal symptoms and food intolerance to characterize the proportion of patients with LGS. MATERIAL AND METHODS: In an observational study, we evaluated 85 patients with functional gastrointestinal symptoms and food intolerance. Gastrointestinal symptoms were classified according to Rom IV into functional abdominal pain (FAP), irritable bowel syndrome (IBS), irritable bowel syndrome diarrhea dominant (IBS-D), irritable bowel syndrome constipation dominant (IBS-C), irritable bowel syndrome with mixed stool (IBS-M), functional abdominal bloating (FAB), functional diarrhea (FD) and unclassified (NC). During eCLE, spontaneous transfer of intravenously applied fluorescein into duodenal lumen (LGS) and following duodenal food challenge (DFC) were analyzed. Blood analysis comprised parameters of mast cell function, histology of duodenal mucosal biopsies analysis of mucosal inflammation, intraepithelial lymphocytes (IELs) as well as number, distribution and morphology of mast cells. RESULTS: 24 patients (9 IBS, 9 FAP, 3 FAB, 1 FD, 2 NC), showed LGS, 50 patients (14 IBS-D, 4 IBS-C, 3 IBS-M, 23 FAP, 3 FAB, 3 NC) had no LGS but responded to DFC and 11 patients (6 NC, 3 FAP, 1 FAB, 1 FD) had no LGS and no response to DFC. The proportion of subgroups with/or without spontaneous leakage of fluorescein (+LGS/-LGS) were IBS-LGS/IBS+LGS 67%/33%, FAP-LGS/FAP+LGS 72%/28%,FAB-LGS/FAB+LGS 50%/50%, NC-LGS/NC+LGS 60%/40%. Subgroup analysis revealed no significant differences for all parameters tested. CONCLUSION: As a proof of concept, the results of our study indicate that eCLE is a clinical useful tool to evaluate patients with disorders of the gut brain axis and those suspicious of LGS. However, the clinical significance of LGS remains unclear. The study should be an incentive to perform a randomized study including healthy controls.

Molecular pathology as a diagnostic aid in difficult-to-classify melanocytic tumours with spitzoid morphology.

Accurate classification of melanocytic proliferations has important implications for prognostic prediction, treatment and follow-up. Although most melanocytic proliferations can be accurately classified using clinical and pathological criteria, classification (specifically distinction between nevus and melanoma) can be challenging in a subset of cases, including those with spitzoid morphology. Genetic studies have shown that mutation profiles differ between primary melanoma subtypes and Spitz nevi. These differences may aid in distinguishing benign from malignant in some melanocytic tumours. Here, we present a selection of melanocytic proliferations with equivocal histopathological criteria, wherein genetic analysis was requested to help guide classification. In two of four cases, the genetic results offered valuable insights, allowing a definitive diagnosis, indicating the diagnostic value of mutation profiling in a real-world routine clinical setting. Although histopathological assessment remains decisive in melanocytic proliferation classification, we recommend including genetic profiling in cases of borderline or atypical lesion to support accurate classification.

Verrucous carcinoma of the esophagus - a case report and literature review.

BACKGROUND: Verrucous carcinoma of the esophagus is a rare disease leading to dysphagia, chest pain, and weight loss. The diagnosis is difficult because even repeated biopsies are often without tumor evidence. We present a patient with verrucous carcinoma of the esophagus and a literature review. CASE REPORT: A 64-year-old patient with dysphagia and sore throat received esophagogastroduodenoscopy illustrating segmental circumferential verrucous inflammation and Candida esophagitis in the middle part of the esophagus. Repeated mucosal biopsies revealed reactive hyperkeratosis of the squamous epithelium with minimal atypia but without ulcera, eosinophilic esophagitis, or suspicion of cancer. Mucosal infection with adenovirus, herpes simplex virus 1, human papilloma virus types, and cytomegaly virus was ruled out. Veruccous carcinoma was detected finally by endoscopic mucosal resection. The patient was successfully treated by esophageal resection. Tumor stage was G1, pT1b, pN0, L0, V0, Pn0, R0. CONCLUSION: The results suggest that macroscopic suspicion of verrucous esophageal carcinoma should lead to resections of larger tissue specimens by EMR to confirm the diagnosis.

Eosinophilic esophagitis and achalasia - just a coincidence?

BACKGROUND: Eosinophilic esophagitis (EoE) is detected frequently in dysphagia and noncardiac chest pain. Management of patients with EoE may be complicated because EoE is associated frequently with esophageal motility disorders. We present the rare case of esophageal achalasia (EA) associated with eosinophilic infiltration and a literature review. MATERIAL AND METHODS: A patient with dysphagia and eosinophilic infiltration referred to our clinic underwent standardized diagnostic work-up including symptom questionnaire, esophagogastroduodenoscopy (EGD) with esophageal biopsies, barium swallow, high-resolution esophageal manometry, and combined intraluminal 24-hour pH-impedance testing (pH/MII). RESULTS: The patient had an Eckardt score of 8. EGD and mucosal biopsies showed typical EoE with > 15 eosinophil leucocytes per high-power field. Barium swallow revealed typical sign of achalasia. HREM indicated EA type 2 according to the Chicago classification. PH/MII was normal. Oral and systemic corticoid therapy were without effect. After successful treatment by pneumatic dilation of the cardia, symptoms relieved and eosinophilic infiltration returned to normal. CONCLUSION: The results suggest that the patient had primary EA associated with eosinophilic infiltration and that the combined occurrence of these rare diseases is not just a coincidence.

Generation of highly differentiated BHY oral squamous cell carcinoma multicellular spheroids.

Three-dimensional (3D) multicellular spheroids (MCS) are considered suitable models in cancer research and anticancer drug development. Although studying the complex tumour characteristics from all different degrees of malignancy is vital, MCS generation has only been described in a few moderately- and poorly differentiated oral squamous cell carcinoma (OSCC) cell lines. No previous study has demonstrated the MCS formation in a highly differentiated OSCC cell line. For the first time, the present study aimed to generate MCS from the highly differentiated OSCC cell line BHY. BHY spheroids were grown in three independent experiments in 96-well plates through the use of the liquid overlay technique. Although BHY cells are grow slowly and are difficult to culture, they formed compact MCS within 24 h. After 3 days of incubation, no further increase in spheroid size was observed. MCS were harvested, paraffin-embedded and 2 µm tissue sections were prepared for further analysis. The diameter and volume of each spheroid were determined. BHY MCS diameter ranged between 46.76 and 233.26 µm, with a volume range from 5.35×104-6.65×106 µm3. In conclusion, using the liquid overlay technique, the highly differentiated OSCC cell line BHY forms different sized spheroids, which may be used for further investigations.

High mast cell density indicates a longer overall survival in oral squamous cell carcinoma.

This study evaluates the effects of tumour-associated mast cells on the prognosis of patients suffering from oral squamous cell carcinoma (OSCC). Tryptase-positive (MCT+) and CD117-positive (CD117+) mast cells were immunohistochemically evaluated in tissue samples of 118 OSCC patients. Besides, various clinicopathological parameters, the influence of the MCT+ and CD117+ mast cell density on overall survival and the incidence of first local recurrence was analysed by Cox regression and competing risk regression. Among all investigated parameters, multiple Cox regression revealed a significant influence of the MCT+ (cut-off at 14.87 mast cells/mm2 stroma; p = 0.0027) and CD117+ mast cell density (cut-off at 33.19 mast cells/mm2 stroma; p = 0.004), the age at primary diagnosis, and the T and N stage (all p-values < 0.05) on overall survival. Patients with a low mast cell density showed a significantly poorer overall survival rate compared to those with a high mast cell density in the tumour-associated stroma. Competing risk regression revealed a significant influence of the resection status (R) on the incidence of first local recurrence (p = 0.0023). A high mast cell density in the tumour-associated stroma of oral squamous cell carcinoma indicates a longer patient survival.

Epigenetic modification suppresses proliferation, migration and invasion of urothelial cancer cell lines.

Epigenetic approaches offer additional therapeutic options, including apoptosis induction, modification of cell cycle regulating proteins and the re-expression of pharmaceutical targets, such as hormone receptors. The present study analyzed the effect of the epigenetic modifiers 5-aza-2'-deoxycytidine and Trichostatin A on the proliferative, migratory and invasive behavior of four urinary bladder cancer cell lines (RT-4, RT-112, VMCUB-1 and T-24), and the expression of various matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). Cell proliferation, migration and invasion assays revealed that treatment with the two epigenetic modifiers resulted in proliferation inhibition in all cell lines, and migration and invasion inhibition in RT-4, RT-112 and T-24 cell lines. Quantitative polymerase chain reaction demonstrated that the mRNA expression of a broad selection of MMPs and their TIMPs was induced in all cell lines, and MMP-14 mRNA expression was suppressed in all cell lines, with the exception of RT-4. In conclusion, epigenetic modifications suppressed the motility and invasiveness of three out of four urothelial cancer cell lines. The inhibitory effect on cell motility appears to be crucial for reduced invasive properties. However, even a broad spectrum of mRNA analysis does not sufficiently explain the loss of invasiveness, as it does not allow for functional conclusions. Further complex urothelial tumour models should be applied to investigate whether epigenetic therapeutic approaches may be an option in urothelial cancer.

Connexin subtype expression during oral carcinogenesis: A pilot study in patients with oral squamous cell carcinoma.

Gap junctional intercellular communication (GJIC) and connexin (Cx) expression were reported in association with carcinogenesis in various types of tumours. In an earlier histomorphometric study, the protein levels of Cx subtypes 26, 43 and 45 were differentially expressed in oral squamous cell carcinoma (OSCC), corresponding lymph node metastases and dysplasia-free oral mucosa. Moreover, membrane Cx43 acted as an independent prognostic marker in OSCC tissues. This study aimed to confirm the expression of described Cx subtypes at the mRNA level. Hence, a reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of Cx26, Cx43 and Cx45 gene expressions was performed in paired carcinoma and mucosa samples of 15 OSCC patients. Additionally, we assessed the interaction between Cx subtype expression and clinicopathological routine parameters. The RT-qPCR analysis revealed that Cx26 was downregulated in OSCC (P=0.01), while Cx43 was marginally upregulated in cancer tissue (P=0.04). Cx45 was significantly overexpressed in OSCC tissue compared with the intraoral mucosa controls (P<0.01), and remained unchanged at different tumour stages. No significant interactions between differential Cx subtype expression and clinicopathological routine parameters were observed. In conclusion, Cx regulation at the transcriptional level appears to be an early event during the initiation and development of OSCC, and is maintained during further progression. However, the mRNA-protein correlation is variable. This may be indicative of post-transcriptional, translational and degradation regulations being associated with the determination of Cx protein concentration during oral carcinogenesis.

Leupaxin is expressed in mammary carcinoma and acts as a transcriptional activator of the estrogen receptor α.

Leupaxin belongs to the group of paxillin proteins and was reported to play a major role in the invasion and migration of prostate cancer cells. In the present study we were able to show by using a cDNA cancer profiling array that leupaxin is upregulated in breast and endometrial cancer, whereas downregulation of leupaxin was observed in lung cancer. In addition, immunohistochemical studies using a leupaxin-specific antibody on human breast cancer specimens (n=127) revealed that leupaxin is expressed mainly in invasive ductal carcinomas and ductal carcinoma in situ (40 and 49% respectively), and only in a minority of lobular mammary carcinomas. To further investigate the role of leupaxin in the progression of breast cancer the expression of leupaxin was analysed in six breast cancer cell lines. The estrogen receptor α (ERα)-positive HCC70 and the ERα-negative MDA-MB­231 cells showed leupaxin expression on the RNA and protein level. Leupaxin localizes in these mammary carcinoma cells at focal adhesion sites and shuttles between membrane and nucleus via its LD4 motif as major nuclear export signal. Interaction partners of leupaxin in the nucleus represent the estrogen receptors ERα and ERß. Both ERα and ERß bind to the LIM domains of leupaxin via their AF-1/DNA binding domains. Furthermore, leupaxin is able to induce transcriptional activity of ERα independent of the presence of estradiol. The specific downregulation of leupaxin expression using siRNAs in mammary carcinoma cells resulted in reduced migratory capability and diminished invasiveness whereas no effect on proliferation was observed. Collectively, these results show that leupaxin has particular influence on the progression and invasion of breast cancer cells and may therefore represent an interesting candidate protein for diagnosis and therapeutic interventions.

Membrane connexin 43 acts as an independent prognostic marker in oral squamous cell carcinoma.

The aim of the present study was to evaluate the expression and localization of connexin (Cx) 26, -43 and -45 in a group of 35 patients with primary oral squamous cell carcinoma (OSCC) with the objective of making a more accurate disease prognosis. We analysed the expression of connexins in tissue samples of primary OSCC, matching oral mucosa free of dysplasia, and its associated lymph node metastases (LNM) by semi-quantitative immunohistochemistry of membrane, cytoplasmic and nuclear connexin expression. The levels of expression were correlated with the overall survival time (OS). Cx43 was overexpressed in tumour cells compared to epithelia in dysplasia-free mucosa. High membrane expression of Cx43 on tumour cells was the only statistically significant and independent prognostic factor of short OS (P=0.0088). Membrane expression of Cx43 in matching dysplasia-free mucosa acted similarly, but did not reach statistical significance (P=0.059). No correlation was found between the Cx26, Cx45 expression and OS. We conclude that Cx43 expression in dysplasia-free mucosa may indicate a very early stage of tumour promotion. Although overexpression of Cx43 is found in invasive tumours we only found membrane Cx43 expression to correlate with OS. This observation suggests that cytoplasmic Cx43 serves as storage and only membrane translocation may promote the formation of gap junctions and gap junctional intercellular communication (GJIC) with prognostic relevance.

Tumor-associated macrophages are involved in tumor progression in papillary renal cell carcinoma.

Tumor-associated macrophages (TAMs) play a key role in cancer development. Especially, the immunosuppressive M2 phenotype is associated with increased tumor growth, invasiveness and metastasis. The differentiation of macrophages to the alternative phenotype M2 is mediated, inter alia, by macrophage colony-stimulating factor (M-CSF). Papillary renal cell carcinoma (RCC) represents a rare tumor type which, based upon histological criteria, can be subdivided into two subtypes (I and II), of which type II is associated with poor prognosis. In both subtypes, typically, a dense infiltrate of macrophages is found. In the present study, the expression of CD68, CD163, M-CSF, Ki-67, and CD31 was examined in 30 type I and 30 type II papillary RCCs (n = 60). Both types of papillary RCCs contained an equally dense infiltrate of CD68-positive macrophages. Nearly all macrophages in papillary RCC type II expressed CD163, a characteristic for M2 macrophages. In type I papillary RCC, less than 30 % of macrophages expressed CD163. Furthermore, tumor cells in type II papillary RCC expressed significantly more M-CSF and showed increased (Ki-67 expression defined) proliferative activity in comparison with type I papillary RCC. In addition, the (CD31 defined) capillary density was higher in type II than in type I papillary RCC. A dense infiltrate of M2 phenotype TAM and high M-CSF expression in tumor cells are key features of type II papillary RCC. These findings might explain why the prognosis of papillary RCC type II is worse than that of type I.

Macrophage-tumor cell interactions regulate the function of nitric oxide.

Tumor cell-macrophage interactions change as the tumor progresses, and the generation of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) plays a major role in this interplay. In early stages, macrophages employ their killing mechanisms, particularly the generation of high concentrations of NO and its derivative reactive nitrogen species (RNS) to initiate tumor cell apoptosis and destroy emerging transformed cells. If the tumor escapes the immune system and grows, macrophages that infiltrate it are reprogramed in situ by the tumor microenvironment. Low oxygen tensions (hypoxia) and immunosuppressive cytokines inhibit iNOS activity and lead to production of low amounts of NO/RNS, which are pro-angiogenic and support tumor growth and metastasis by inducing growth factors (e.g., VEGF) and matrix metalloproteinases (MMPs). We review here the different roles of NO/RNS in tumor progression and inhibition, and the mechanisms that regulate iNOS expression and NO production, highlighting the role of different subtypes of macrophages and the microenvironment. We finally claim that some tumor cells may become resistant to macrophage-induced death by increasing their expression of microRNA-146a (miR-146a), which leads to inhibition of iNOS translation. This implies that some cooperation between tumor cells and macrophages is required to induce tumor cell death, and that tumor cells may control their fate. Thus, in order to induce susceptibility of tumors cells to macrophage-induced death, we suggest a new therapeutic approach that couples manipulation of miR-146a levels in tumors with macrophage therapy, which relies on ex vivo stimulation of macrophages and their re-introduction to tumors.

N-cadherin expression in malignant germ cell tumours of the testis.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18-35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth. METHODS: Tumour tissues were acquired from 113 male patients and investigated by immunohistochemistry, as were the three TGCT cell lines NCCIT, NTERA-2 and Tcam2. A monoclonal antibody against N-cadherin was used. RESULTS: Tumour-free testis and intratubular germ cell neoplasias (unclassified) (IGCNU) strongly expressed N-cadherin within the cytoplasm. In all seminomas investigated, N-cadherin expression displayed a membrane-bound location. In addition, the teratomas and yolk sac tumours investigated also differentially expressed N-cadherin. In contrast, no N-cadherin could be detected in any of the embryonal carcinomas and chorionic carcinomas examined. This expression pattern was also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma. CONCLUSIONS: N-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT.

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells.

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)­resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT­resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.