Expression of L1CAM, COX-2, EGFR, c-KIT and Her2/neu in anaplastic pancreatic cancer: putative therapeutic targets?

AIMS: Undifferentiated (anaplastic) pancreatic cancer and undifferentiated pancreatic carcinoma with osteoclast-like giant cells (giant cell tumour) are rare variants of pancreatic ductal adenocarcinoma. Representing biologically highly aggressive neoplasms, they are frequently diagnosed at an advanced stage. The response to established chemo- or radiochemotherapeutic treatment regimens is poor, and undifferentiated pancreatic cancer generally has a dismal prognosis. As additional therapeutic options have not yet been investigated in undifferentiated pancreatic cancer, the aim was to analyse the expression of putative therapeutic targets that have shown promising results in various other neoplasms. METHODS AND RESULTS: Fifteen cases of undifferentiated pancreatic cancer (seven containing osteoclast-like giant cells) were investigated clinicopathologically and immunohistochemically for putative therapeutic targets. Whereas L1CAM, cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR) were found to be significantly expressed in 80%, 93% and 87% of the investigated tumours, respectively, there was no substantial expression of c-kit (CD117) and there was no detectable expression of Her2/neu. CONCLUSIONS: The expression of L1CAM, COX-2 and EGFR in the majority of undifferentiated pancreatic carcinomas suggests that they might represent targets for adjuvant therapy in anaplastic pancreatic cancer. On the other hand, c-kit and Her2/neu seem to have no relevance for the therapy of these tumours.

Expression pattern and functional relevance of epidermal growth factor receptor and cyclooxygenase-2: novel chemotherapeutic targets in pancreatic endocrine tumors?

OBJECTIVES: Pancreatic endocrine tumors represent morphologically and biologically heterogeneous neoplasms. Well-differentiated endocrine tumors (benign or of uncertain behavior) can be distinguished from well-differentiated and poorly differentiated endocrine carcinomas. Although many well-differentiated endocrine carcinomas show rather low rates of tumor growth, more than two-thirds of pancreatic endocrine carcinomas display distant metastases at the time of diagnosis. As the currently applied therapies beyond surgery only achieve partial or complete response rates of approximately 15%, additional chemotherapeutic targets are needed, especially in the therapy of inoperable and progressive pancreatic endocrine carcinomas. METHODS: The expression of epidermal growth factor receptor (EGFR) and cyclooxygenase (COX)-2 were investigated in 110 clinically and pathomorphologically well-characterized pancreatic endocrine tumors, using immunohistochemistry and immunoblot analyses. Functional tests were performed using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line beta-TC-3. RESULTS: The expression of EGFR correlated significantly with the grade of malignancy, increasing from low rates of expression in benign tumors and tumors of uncertain behavior to high rates of expression in well- and poorly differentiated endocrine carcinomas. The expression of COX-2 was independent of the malignant potential, but was more frequently expressed in primary tumors than in metastases. The treatment of the human pancreas carcinoid cell line BON and the mouse insulinoma cell line beta-TC-3 with EGFR and COX-2 inhibitors (monotherapy and combined therapy) resulted in a significant, dose-dependent reduction of cell viability coupled with increased apoptosis. CONCLUSIONS: Our results suggest that EGFR and COX-2 may represent useful additional chemotherapeutic targets in pancreatic endocrine tumors.

Recurrent deletions at 6q in early age of onset non-HNPCC- and non-FAP-associated intestinal carcinomas. Evidence for a novel cancer susceptibility locus at 6q14-q22.

Hereditary intestinal cancers mainly occur in the framework of the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) syndromes. However, in about 50% of young patients with intestinal cancer clinically suspicious of an inherited cancer predisposition, no underlying molecular alteration can be identified. To determine the genetic profile of early onset intestinal cancer in the absence of a known cancer predisposition, we have analyzed 12 small and large intestinal tumors from 11 non-FAP, non-HNPCC, and noninflammatory bowel disease patients diagnosed under the age of 35years using a combination of comparative genomic hybridization and loss of heterozygosity (LOH) analysis. The distribution of chromosomal gains and deletions was similar to late onset carcinomas except for 6q alterations which were found in 50% of carcinomas. To define a shared region affected by allelic imbalance, detailed LOH analysis at chromosome 6 was performed on three carcinomas from two patients which showed small interstitial 6q deletions. A minimal area of deletion overlap between markers D6S1652 and D6S1657 (6q14-22) was identified. In a patient with small and large intestinal carcinoma and seven adenomas, loss of identical parental 6q14-22 alleles was seen in both carcinomas and in three of the adenomas. Our data indicate that alterations affecting 6q are frequent in early onset intestinal carcinomas. Moreover, deletions of identical parental alleles in a 35 Mbp area at 6q in multiple independent tumors from one of the patients are compatible with the existence of a novel 6q-associated cancer susceptibility syndrome.

Establishment, characterization and drug sensitivity testing in primary cultures of human thymoma and thymic carcinoma.

Thymomas and thymic carcinomas are peculiar epithelial tumors of the anterior mediastinum. They may show aggressive clinical behavior and are a paradigm for the interaction between the tumor and the immune system. So far, adequate functional studies enabling a better understanding of this malignancy have not been performed, since human thymoma/thymic carcinoma cell lines have not been available. Here, the authors describe the establishment, characterization and functional analyses of epithelial cell lines from a Type B1-thymoma and a poorly differentiated thymic carcinoma. By Fluorescence-activated cell sorting (FACS) analyses, both cell lines were aneuploid. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array-based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX-2 inhibitor reduced cell viability in both cell lines in a dose-dependent manner. In conclusion, these first cell lines of a thymoma and a CD5-positive thymic carcinoma are useful tools for further in vitro studies of cellular, molecular and genetic aspects of the disease and for functional tests to evaluate new therapeutic targets.

A mathematical approach predicting the number of events in different tumors.

Supported by different investigations, multi-step models for tumorigenesis have been proposed for epithelial tumors. The age specific incidence of some cancers shows an exponential rise with increasing patient age. Yet, the onset and the slope of incidence curves varies between tumor types. One simple explanation for this disparity is that the number of mutations required for transformation differs in various tissues. We used a homogeneous Poisson process to estimate the number of events (N) and the intensity or event rate (lambda) that might be needed for cancer development in various tissues (colon, prostate, oralpharynx, larynx). Estimations were performed, including 95% confidence intervals, for the male and female population. The expected number of events needed was higher in adenocarcinomas (colorectal carcinoma: N approximately 10 for females and N approximately 11.0 for males; prostatic cancer: N approximately 23) than in squamous cell carcinomas (oropharynx: N approximately 5-6 for females and N approximately 6 for males; larynx: N approximately 7 for females and N approximately 8 males). Still, alternative models fixing N to values within the 95% confidence intervals determined, showed good coincidence with epidemiological data. Although the herein applied mathematical model neglects several biologic conditions, especially a presumed acceleration of mutation rates after tumor initiation it offers a plausible theory for the given epidemiologic data and matches with molecular biologic findings in the investigated cancers.

Cyclooxygenase-2 inhibition induces apoptosis signaling via death receptors and mitochondria in hepatocellular carcinoma.

Inhibition of cyclooxygenase (COX)-2 elicits chemopreventive and therapeutic effects in solid tumors that are coupled with the induction of apoptosis in tumor cells. We investigated the mechanisms by which COX-2 inhibition induces apoptosis in hepatocellular carcinoma (HCC) cells. COX-2 inhibition triggered expression of the CD95, tumor necrosis factor (TNF)-R, and TNF-related apoptosis-inducing ligand (TRAIL)-R1 and TRAIL-R2 death receptors. Addition of the respective specific ligands further increased apoptosis, indicating that COX-2 inhibition induced the expression of functional death receptors. Overexpression of a dominant-negative Fas-associated death domain mutant reduced COX-2 inhibitor-mediated apoptosis. Furthermore, our findings showed a link between COX-2 inhibition and the mitochondrial apoptosis pathway. COX-2 inhibition led to a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, followed by translocation of Bax to mitochondria and cytochrome c release from mitochondria. Consequently, overexpression of Mcl-1 led to inhibition of COX-2 inhibitor-mediated apoptosis. Furthermore, blocking endogenous Mcl-1 function using a small-interfering RNA approach enhanced COX-2 inhibitor-mediated apoptosis. It is of clinical importance that celecoxib acted synergistically with chemotherapeutic drugs in the induction of apoptosis in HCC cells. The clinical relevance of these results is further substantiated by the finding that COX-2 inhibitors did not sensitize primary human hepatocytes toward chemotherapy-induced apoptosis. In conclusion, COX-2 inhibition engages different apoptosis pathways in HCC cells stimulating death receptor signaling, activation of caspases, and apoptosis originating from mitochondria.

Mutational status of the epidermal growth factor receptor (EGFR) gene in thymomas and thymic carcinomas.

Epithelial tumours of the thymus (thymoma, thymic carcinoma) are rare tumours of the anterior mediastinum. Current treatment options of advanced stage thymomas and thymic carcinomas include a multimodal therapy with radio- and chemotherapy as well as surgery. In recent years, new therapeutic targets such as EGFR (epidermal growth factor receptor), COX-2 and KIT have emerged as new potential therapeutic targets. So far, EGFR mutational status of different subtypes of epithelial tumours of the thymus has been analyzed only inappropriately. We have investigated 20 different subtypes of thymomas (type A, AB, and B3) and thymic carcinomas for mutations in exons 18, 19, 20, and 21 of the EGFR gene and performed immunohistochemistry for EGFR. Concerning immunohistochemistry, most of the cases (17/20) had a strong positive staining. Although sequence alterations were found in four samples, none of these alterations led to amino acid changes in the tyrosine kinase domain of EGFR comparable to those in non-small cell lung cancer. Thus EGFR-expression in thymic tumours does not rely on mutations in critical functional (activation) domains of the EGFR-gene. Experimental and therapeutic approaches have to consider this difference.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma: correlation with microvessel density.

AIM: Cyclooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins. Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms. The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis, tumorigenesis and inflammatory processes. This study was to clarify their role in pancreatic adenocarcinomas. METHODS: We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocarcinomas of different grade and stage. The results were compared with microvessel density and clinicopathological data. RESULTS: Twenty-one (52.5%) of the cases showed iNOS expression, 15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors. Staining intensity was different between the tumors. No correlation between iNOS and COX-2 expression was seen. There was no relationship with microvessel density. However, iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression. There was no correlation with other clinicopathological data. CONCLUSION: Approximately half of the cases expressed iNOS and COX-2. These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas. Due to a low prevalence of COX-2 expression, chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

KRAS, EGFR, PDGFR-α, KIT and COX-2 status in carcinoma showing thymus-like elements (CASTLE).

BACKGROUND: CASTLE (Carcinoma showing thymus-like elements) is a rare malignant neoplasm of the thyroid resembling lymphoepithelioma-like and squamous cell carcinoma of the thymus with different biological behaviour and a better prognosis than anaplastic carcinoma of the thyroid. METHODS: We retrospectively investigated 6 cases of this very rare neoplasm in order to investigate the mutational status of KRAS, EGFR, PDGFR-α and KIT, as well as the immunohistochemical expression pattern of CD117, EGFR and COX-2, and possibly find new therapeutic targets. RESULTS: Diagnosis was confirmed by a moderate to strong expression of CD5, CD117 and CK5/6, whereas thyroglobulin, calcitonin and TTF-1 were negative in all cases. Tumors were also positive for COX-2 and in nearly all cases for EGFR. In four cases single nucleotide polymorphisms (SNPs) could be detected in exon 12 of the PDGFR-α gene (rs1873778), in three cases SNPs were found in exon 20 of the EGFR gene (rs1050171). No mutations were found in the KIT and KRAS gene. CONCLUSIONS: All tumors showed a COX-2 expression as well as an EGFR expression except for one case and a wild-type KRAS status. No activating mutations in the EGFR, KIT and PDGFR-α gene could be detected. Our data may indicate a potential for targeted therapies, but if these therapeutic strategies are of benefit in CASTLE remains to be determined. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1658499296115016.

Expression and therapeutic relevance of heat-shock protein 90 in pancreatic endocrine tumors.

Pancreatic endocrine tumors (PET) represent a heterogenous group of neoplasms. Although surgical resection is considered a safe and effective treatment for many PET, therapeutic options for inoperable and progressive PET are limited. The expression of heat-shock protein (HSP) 90 was investigated in 120 clinically and pathomorphologically well-characterized PET from 84 patients using immunohistochemistry. In addition, in 19 snap-frozen PET and in three healthy pancreatic tissues, we performed immunoblot analyses, and in 15 snap-frozen PET and in three healthy pancreatic tissues, we investigated the expression of HSP90 isoforms by means of semiquantitative RT-PCR. Functional tests were conducted using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line ß-TC-3. HSP90 was expressed in 95% of the PET patients. The transcript levels of the HSP90 isoforms HSP90α, HSP90ß, glucose-related protein 94, and TNF receptor-associated protein 1 were significantly increased in PET compared with non-neoplastic pancreatic tissues. The treatment of the cell lines BON and ß-TC-3 with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin resulted in significant, dose-dependent reduction of cell viability, cell cycle arrest, and increased apoptosis. Furthermore, HSP90 inhibition induced the degradation and inactivation of several oncogenetic HSP90 client proteins in a time- and dose-dependent manner. HSP90 inhibitors increased the therapeutic effects of doxorubicin and 5-fluorucacil in BON and ß-TC-3 cells. HSP90 is expressed in the vast majority of PET and its inhibition reveals significant treatment effects in vitro. Thus, HSP90 qualifies as a promising new target.

Fundamental differences in the neural invasion behavior of pancreatic endocrine tumors: relevance for local recurrence rates?

Neural invasion represents an important prognostic factor in pancreatic cancer, and it is thought to be one of the main causes for the high rate of postoperative local recurrences in pancreatic ductal adenocarcinomas. In contrast to the latter, systematic investigations of the mode and extent of neural invasion in pancreatic endocrine tumors have not yet been carried out, although this process represents an important feature in the classification of these tumors. In the present study, a total of 48 pancreatic endocrine tumors were analyzed including 10 well-differentiated endocrine tumors of uncertain behavior, 33 well-differentiated endocrine carcinomas, and 5 poorly differentiated endocrine carcinomas. Neural invasion was found in a large subset (73%) of pancreatic endocrine tumors. The frequency of neural invasion correlated with the grade of malignancy but occurred irrespective of functional activity, hormone phenotype, or histomorphology. Analogous to pancreatic ductal adenocarcinoma, the expression of epidermal growth factor receptor and nerve growth factor, which were expressed in 50% and 100% of the tumors, respectively, seemed to be associated with the frequency of neural invasion. However, in contrast to pancreatic ductal adenocarcinoma, neural invasion in pancreatic endocrine tumors was only detected within the tumor boundaries and did not reach beyond the tumor invasion front. This phenomenon may explain the low rate of local relapses after tumor resection in pancreatic endocrine tumors despite the high frequency of neural invasion.

Expression and mutational status of PDGFR in thymic tumours.

BACKGROUND: There is an ongoing search for new therapeutic targets in invasive non-resectable thymic tumours because of the low response rates in current chemotherapeutic treatment modalities. In this study, the possibility that platelet-derived growth factor receptor A (PDGFRA) and/ or PDGFRB may represent potential therapeutic targets in epithelial tumours of the thymus was investigated. PATIENTS AND METHODS: Tissue samples were obtained by thymectomy from 36 different patients with epithelial tumours of the thymus (26 thymomas types A, AB, B1-3 and 10 thymic carcinomas). Normal thymi from three young children were used as controls. The PDGFRA and PDGFRB protein expressions as well as the mutational statuses of exons 12, 14 and 18 of the PDGFRA gene were analyzed. RESULTS: All the subtypes of thymomas and the thymic carcinomas showed staining for PDGFRA, but no mutations in the known mutational hotspots were identified. Only about one third of the tumours stained for PDGFRB. PDGFRA and PDGFRB protein staining were slightly positively correlated. CONCLUSION: PDGFRA may represent a potential therapeutic target in thymic tumours.

Ex vivo analysis of antineoplastic agents in precision-cut tissue slices of human origin: effects of cyclooxygenase-2 inhibition in hepatocellular carcinoma.

Cultures of precision-cut tissue slices allow the investigation of substance effects on human tissues under in vivo-like conditions over a limited time span. We have adapted the model for direct analyses of antineoplastic substances on tumor tissues. We have recently demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors strongly suppress growth of human hepatocellular carcinoma (HCC) cells in vitro and nude mouse HCC implants by inducing apoptosis and reducing proliferation. We have now analyzed the effects of COX-2 inhibition on human tumor tissue. Three hundred micrometer slices of tumorous and non-tumorous liver tissue from three surgically resected HCCs were cultured with increasing concentrations of the selective COX-2 inhibitor Meloxicam (20-200 microM) for 6, 12, 24, and 48 h. The cultured tissue slices were analysed morphologically and by immunohistology for proliferation (Ki-67), apoptosis (M30), and COX-2 expression. COX-2 was expressed in all HCCs and in the non-tumorous liver tissue. Cytoplasmic COX-2 immunoreactivity in HCCs increased during culturing time. In two of three cases, COX-2 inhibition significantly increased tumor cell apoptosis in HCCs, whereas the low basal apoptosis rate in the non-tumorous liver parenchyma did not change. Tumor cell proliferation was mildly reduced, but the changes did not reach statistical significance. These results demonstrate that the precision-cut tissue slice culture model is a useful tool to analyze directly drug-dependent antitumorous or unwanted organ-specific effects. The analysis of COX-2 inhibition lends further support to the antineoplastic effects previously demonstrated in vitro and in animal models.

The role of Epstein-Barr virus in acute and chronic hepatitis.

BACKGROUND/AIMS: Epstein-Barr virus has a seroprevalence of more than 80% world wide and is known to be associated with hepatitis. However, little is known about the underlying pathogenesis and immunmechanisms and no standard diagnostic criteria to diagnose EBV-hepatitis are available. METHODS: We collected liver biopsies (n=21) with the tentative diagnosis of EBV induced hepatitis according to pathological changes and traceable EBV genome by PCR. Correlation with serological data revealed acute in seven cases, convalescent in two cases, past EBV infection in six cases. Viral RNA was visualised by in situ hybridisation within nuclei of lymphocytes. RESULTS: In seven of 68 liver biopsies with the diagnosis 'liver disease of unknown aetiology' EBV genome in the tissue was demonstrated indicating a possible role for EBV in the induction of hepatitis or a trapping of infected lymphocytes within the liver. In a control group of 20 EBV-seropositive patients with steatohepatitis EBV-DNA PCR of the liver tissue was negative. Immunohistochemistry identified CD3 and CD8 positive T-lymphocytes as the main lymphocytic infiltrate in EBV hepatitis. CONCLUSIONS: EBV hepatitis should be taken into consideration in case of typical histopathological changes and a positive DNA PCR of liver biopsy. Serological confirmation of the diagnosis is inevitable.

COX-2 upregulation in thymomas and thymic carcinomas.

The treatment of advanced stage thymomas and thymic carcinomas is a multimodal therapy. New therapeutic targets are currently under investigation, including the epidermal growth factor receptor (EGFR) as well as KIT. A number of studies have shown protumorigenic potential of Cyclooxygenase-2 (COX-2) in a variety of human malignancies, but so far it is unknown whether COX-2 is expressed in primary malignancies of the thymus. Using tissue microarrays, the expression of COX-2, microsomal-PGES-1 and -PGES-2 (mPGES-1 and mPGES-2), as well as EGFR was evaluated in different subtypes of thymoma and thymic carcinomas. COX-2 was expressed in all subtypes as determined by immunohistochemistry. Some cases of type B2 and thymic carcinomas had COX-2 staining levels classified as mild to moderate. However, when measuring the optical color intensity, no significant differences could be detected. Concerning the expression levels, a weak correlation between the expression of COX-2, mPGES-1 and mPGES-2 as well as EGFR was found. Furthermore, additional cases of thymomas and thymic carcinomas were analyzed by COX-2 Western immunoblot analysis and were compared to normal thymi. The analysis showed that thymomas and thymic carcinomas had a significantly stronger COX-2 expression than that of the normal thymi (p < 0.04). In summary, COX-2 is expressed in all subtypes of thymomas and thymic carcinomas and thus represents, in addition to EGFR and KIT, a potential therapeutic target. Further studies are needed in order to determine whether a combined therapy using COX-2 inhibitors in addition to the evolving anti-EGFR antibody therapy may be considered as a treatment option.

Mixed hepatoblastoma in an adult.

We report a case in an elderly adult of a highly malignant liver tumor with blastoid features that resembled hepatoblastoma. A liver tumor with a diameter of 23 cm was removed in a 78-year-old woman. The tumor showed highly differentiated epithelial hepatocellular and poorly differentiated epithelial and mesenchymal components. The blastoid nature and pluripotent differentiation potential were supported by immunohistologic analysis and suggest an origin of a poorly differentiated pluripotent hepatic cell with the potential to mature. We believe that this case of a mixed hepatoblastoma in an adult should be added to the growing number of presumed hepatic precursor cell neoplasms in adults.

Proliferative activity of microvascular cells in glioblastomas does not correlate with time to recurrence.

The time to recurrence operation (TR) is a good growth parameter, in particular for glioblastomas. Recently, we have shown that Ki-67 labeling index (LI) of tumor cells has a high inverse correlation with this time interval. In the current study, the LI of microvascular cells (MVC) was examined in the same glioblastoma cases. The LI of MVC of primary and recurrent tumors had no relationship and did not show any correlation to TR. The growth fraction of MVC was significantly lower than that of tumor cells. The MVC in glioblastomas seems to have chaotical proliferation properties without any link to the tumor growth potential. This observation may have implications for anti-angiogenic therapy.