Gerhard Dekan

Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells

Basic fibroblast growth factor (FGF‐2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF‐2 and FGF receptors (FGFR‐1 to ‐4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF‐2 and FGFR in vitro and in vivo. FGF‐2 expression did correlate with a short doubling time as well as with potent anchorage‐independent growth of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF‐2 into the extracellular space. Expression levels of FGFR‐1 and ‐2 in NSCLC cell lines correlated with FGF‐2 production. FGFR were located at the plasma membranes in some low FGF‐2‐producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF‐2 (rFGF‐2). In NSCLC cell lines with an enhanced FGF‐2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF‐2 and growth inhibition by FGF‐2‐neutralising antibodies. In contrast, several agents antagonised FGF‐2 intracellularly impaired growth of almost all NSCLC cell lines. Our data suggest a role of FGF‐2 and FGFR in the growth stimulation of NSCLC cells possibly via an intracrine mechanism. Int. J. Cancer 83:415–423, 1999.

Long-Lived Th2 Memory in Experimental Allergic Asthma

Although life-long immunity against pathogens is beneficial, immunological memory responses directed against allergens are potentially harmful. Because there is a paucity of information about Th2 memory cells in allergic disease, we established a model of allergic asthma in BALB/c mice to explore the generation and maintenance of Th2 memory. We induced disease without the use of adjuvants, thus avoiding Ag depots, and found that unlike allergic asthma in mice immunized with adjuvant, immunizing with soluble and aerosol OVA resulted in pathological lung lesions resembling human disease. To test memory responses we allowed mice with acute disease to recover and then re-exposed them to aerosol OVA a second time. Over 400 days later these mice developed OVA-dependent eosinophilic lung inflammation, airway hyperresponsiveness, mucus hypersecretion, and IgE. Over 1 year after recuperating from acute disease, mice had persistent lymphocytic lung infiltrates, Ag-specific production of IL-4 and IL-5 from spleen and lung cells in vitro, and elevated IgG1. Moreover, when recuperated mice were briefly aerosol challenged, we detected early expression of Th2 cytokine RNA in lungs. Taken together, these data demonstrate the presence of long-lived Th2 memory cells in spleen and lungs involved in the generation of allergic asthma upon Ag re-exposure.

The TP53 genotype but not immunohistochemical result is predictive of response to cisplatin-based neoadjuvant therapy in stage III non-small cell lung cancer.

BACKGROUND The cytotoxic effects of cisplatin and anthracyclins have been attributed to apoptosis induction, which has been recognized as a major function of the TP53 gene. The TP53 gene appears to be mutated in about 50% of cases of non-small cell lung cancer. A possible dependence of chemotherapy response on TP53 genotype was evaluated retrospectively in a group of patients with advanced non-small cell lung cancer and induction treatment. METHODS Patients with complete or partial remission were compared with those with stable or progressive disease with respect to TP53 genotype and overall survival. Mutations in the TP53 gene were detected by complete direct sequencing (exons 2-11). RESULTS A normal TP53 genotype proved to be significantly associated with major response to chemotherapy (P <.001). Overall, no association was found between p53 protein expression and TP53 genotype. A normal TP53 genotype was found to be highly sensitive in predicting response to treatment, whereas a mutant genotype was revealed to be specific in predicting lack of response. The difference in overall length of survival was significant between patients exhibiting a normal TP53 genotype (corresponding to those whose disease responded to chemotherapy) and patients showing mutant TP53 genotype (corresponding to those who had disease resistant to chemotherapy, P =.027). CONCLUSIONS In a small cohort of patients with advanced non-small cell lung cancer we found a direct link between normal TP53 genotype and response to cisplatin-based induction treatment and also between mutant genotype and resistance to treatment, whereas p53 immunohistochemical result was predictive of neither.

Comparison between mycophenolate mofetil- and azathioprine-based immunosuppressions in clinical lung transplantation.

BACKGROUND The aim of the study was to assess the impact of mycophenolate mofetil (MMF) on the early phase after lung transplantation. PATIENTS AND METHODS Thirty-eight consecutive patients between November 1994 and January 1997 were treated with cyclosporine, prednisolone, antithymocyte globuline induction therapy, and either MMF (n = 21) or azathioprine (Aza) (n = 17). Four patients from the MMF group and 2 patients from the Aza group were intubated and in the ICU prior to transplantation. Demographic data and primary diagnosis were comparable. MMF was administered at a dosage of 2 gm/day whereas Aza was initiated at 2 mg/kg/day and adapted by leukocyte count. Three-month survival and incidence of rejections and infections were compared. RESULTS Six-month survival in the MMF group was 76% compared to 65% in the Aza group (n.s.). The mean number of acute rejection episodes in the MMF and Aza group were 0.29+/-0.10 and 1.53+/-0.29 (p<0.01) respectively. Transbronchial biopsy (TBB) results > or =grade 2 ISHLT were seen in 10% of MMF and in 43% of Aza-treated patients; completely free from rejection were 17 MMF and 3 Aza patients. The mean number of infections per patient in the MMF and Aza group were 1.57+/-0.29 and 2.29+/-0.40 respectively, bacterial (1.10 vs. 1.71), viral (0.35 vs. 0.33), and fungal (0.14 vs. 0.24) infections were the same in both groups. CONCLUSIONS These data result suggest that mycophenolate mofetil therapy is more effective in preventing rejection episodes in patients early after lung transplantation than therapy with azathioprine. We therefore conclude that MMF is a safe and effective drug to optimize immunosuppressive therapy in the early phase after lung transplantation.

Intranasal Treatment with a Recombinant Hypoallergenic Derivative of the Major Birch Pollen Allergen Bet v 1 Prevents Allergic Sensitization and Airway Inflammation in Mice

Background: The major birch pollen allergen Bet v 1 represents one of the most prevalent environmental allergens responsible for allergic airway inflammation. Objective: In the present study we sought to compare the complete recombinant Bet v 1 allergen molecule with genetically produced hypoallergenic fragments of Bet v 1 regarding mucosal tolerance induction in a mouse model of allergic asthma. Methods: BALB/c mice were intranasally treated with recombinant Bet v 1 or with two recombinant Bet v 1 fragments (F I: aa 1–74; F II: aa 75–160) prior to aerosol sensitization with birch pollen and Bet v 1. Results: Intranasal application of F II, containing the major T cell epitope, led to significant reduction of IgE/IgG1 antibody responses, in vitro cytokine production (IL-5, IFN-γ, IL-10) and negative immediate cutaneous hypersensitivity reactions comparable to the pretreatment with the complete rBet v 1 allergen. Moreover, airway inflammation (eosinophilia, IL-5) was inhibited by the pretreatment with either the complete Bet v 1 or F II. However, for prevention of airway hyperresponsiveness the complete molecule was required. The mechanisms leading to immunosuppression seemed to differ in their dependence on the conformation of the molecules, since tolerance induced with the complete Bet v 1, but not with F II, was transferable with spleen cells and associated with increased TGF-β mRNA levels. Conclusion: We conclude that mucosal tolerance induction with recombinant allergens and genetically engineered hypoallergenic derivatives thereof could provide a convenient and safe intervention strategy against type I allergy.

Successful lung transplantation for bronchiolitis obliterans after allogeneic marrow transplantation.

Background. Bone marrow transplantation (BMT) is an established therapy for a variety of hematological diseases with curative potential. However, despite improvements in supportive care, pulmonary complications remain a significant cause of morbidity and mortality. Methods. We report on a patient who received a double lung transplantation (LTX) for therapy-refractory bronchiolitis obliterans (BO) associated with extensive chronic graft-versus-host disease (GVHD) after allogeneic BMT. Results. At present, 38 months after BMT and 23 months after LTX, the patient is in complete hematological and cytogenetic remission and without signs of respiratory distress. Conclusions. This case illustrates that lung transplantation could be a therapeutic option in selected patients with BO after allogeneic BMT that is associated with extensive chronic GVHD and who are refractory to conventional immunosuppressive therapy.

Genome-wide CpG island methylation analyses in non-small cell lung cancer patients

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.

Expression of the multidrug resistance-associated protein (MRP) gene in colorectal carcinomas.

To determine the clinical significance of MRP in patients with colorectal carcinomas, we have studied the expression of the MRP gene by reverse transcription-polymerase chain reaction (RT-PCR) (n = 105) and by immunohistochemistry (n = 30). MRP mRNA expression was observed in 92 (88%) tumour specimens. Positive MRP staining with monoclonal antibodies QCRL-1 and QCRL-3 was detected in all samples studied with strong staining in seven (23%) and weak staining in 23 (77%) specimens. Strong MRP staining in these samples did not appear to be related to the age and sex of the patients, localization of the primary tumour, histological grade, tumour size, lymph node metastasis, distant metastasis and tumour stage. Strong MRP staining was not associated with MDR1 RNA or P-glycoprotein (P-gp) expression. Kaplan-Meier curves revealed that overall survival of patients with strong MRP-staining tumours was similar to the survival of patients with weak-staining tumours. These data indicate that the MRP gene is expressed in primary colorectal carcinomas but is neither related to known prognostic factors nor a prognostic factor by itself.

Aneuploidy of chromosome 7 can be detected in invasive lung cancer and associated premalignant lesions of the lung by fluorescence in situ hybridisation

In the present study the chromosomal status of seven invasive non small cell lung cancer specimens and associated premalignant lesions was investigated. By fluorescence in situ hybridisation (FISH) with centromere specific probes, an increase in the percentage of aneuploid cells from pre-invasive to invasive lesions could be demonstrated (mean 8.5 and 59%, respectively, for chromosome 7). Furthermore, mean chromosome copy numbers were higher in invasive carcinomas as compared to premalignant lesions, indicating polyploidization during tumor development. Increasing evidence suggests that aberrations of chromosome 7 occur early in the development of lung cancer. Whether these aberrations can be used as a biomarker for future neoplastic progression remains to be determined.

Repeated Aerosol Allergen Exposure Suppresses Inflammation in B-Cell-Deficient Mice with Established Allergic Asthma

Background: Repeated allergen administration is a well-established therapeutic strategy for desensitizing patients with allergic disease. Similarly, repeated inhalation of antigen by mice with established allergen-induced asthma suppresses allergic inflammation. The mechanisms underlying antigen-dependent suppression of allergic immune responses remain unknown. In previous studies, we found that repeated aerosol antigen challenges in sensitized mice reduced eosinophils while increasing plasma cells and antibody in the lungs. We sought to test whether plasma cells and antibody played a role in suppression of allergic disease. Methods: We primed wild-type and B-cell-deficient (µMT) mice with 25 µg ovalbumin (OVA) precipitated in alum on days 0 and 5, nebulized weekly with 1% OVA, 1 h, twice daily, for up to 6 weeks, and assessed lung inflammation, mucus hypersecretion, and IgE/IgG1. Results: Kinetic studies revealed that initial aerosol exposure induced high numbers of eosinophils, lymphocytes, and macrophages within lung infiltrates and increased mucus production in wild-type mice. After 3–4 weeks of antigen exposure, eosinophils diminished while lymphocytes, plasma cells, and macrophages and mucus hypersecretion increased. However, by 6 weeks, lung inflammation and mucus hypersecretion were dramatically reduced. In contrast, repeated aerosol challenges maintained OVA-specific IgG1 and IgE production. Repeated aerosol antigen challenges in µMT mice resulted in reduced lung inflammation and mucus hypersecretion and the development of smooth muscle hypertrophy of the pulmonary microvasculature. Conclusions: B cells and antibody do not appear to play a role in antigen-dependent suppression of allergic responses in mice.