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Purpose: It was the aim of our study to establish an extensive panel of non-small cell lung cancer (NSCLC) xenograft models useful for the testing of novel compounds and for the identification of biomarkers.Experimental Design: Starting from 102 surgical NSCLC specimens, which were obtained from primarily diagnosed patients with early-stage tumors (T2/T3), 25 transplantable xenografts were established and used for further investigations.Results: Early passages of the NSCLC xenografts revealed a high degree of similarity with the original clinical tumor sample with regard to histology, immunohistochemistry, as well as mutation status. The chemotherapeutic responsiveness of the xenografts resembled the clinical situation in NSCLC with tumor shrinkage obtained with paclitaxel (4 of 25), gemcitabine (3 of 25), and carboplatin (3 of 25) and lower effectiveness of etoposide (1 of 25) and vinorelbine (0 of 11). Twelve of 25 NSCLC xenografts were >50% growth inhibited by the anti-epidermal growth factor receptor (EGFR) antibody cetuximab and 6 of 25 by the EGFR tyrosine kinase inhibitor erlotinib. The response to the anti-EGFR therapies did not correlate with mutations in the EGFR or p53, but there was a correlation of K-ras mutations and erlotinib resistance. Protein analysis revealed a heterogeneous pattern of expression. After treatment with cetuximab, we observed a down-regulation of EGFR in 2 of 6 sensitive xenograft models investigated but never in resistant models.Conclusion: An extensive panel of patient-derived NSCLC xenografts has been established. It provides appropriate models for testing marketed as well as novel drug candidates. Additional expression studies allow the identification of stratification biomarkers for targeted therapies.

Supplementary Data from Comparative Profiling of the Novel Epothilone, Sagopilone, in Xenografts Derived from Primary Non–Small Cell Lung Cancer

Supplementary Data from Establishment of Patient-Derived Non–Small Cell Lung Cancer Xenografts as Models for the Identification of Predictive Biomarkers

Purpose: Characterization of new anticancer drugs in a few xenograft models derived from established human cancer cell lines frequently results in the discrepancy between preclinical and clinical results. To take the heterogeneity of tumors into consideration more thoroughly, we describe here a preclinical approach that may allow a more rational clinical development of new anticancer drugs.Experimental Design: We tested Sagopilone, an optimized fully synthetic epothilone, in 22 well-characterized patient-derived non–small cell lung cancer models and correlated results with mutational and genome-wide gene expression analysis.Results: Response analysis according to clinical trial criteria revealed that Sagopilone induced overall responses in 64% of the xenograft models (14 of 22), with 3 models showing stable disease and 11 models showing partial response. A comparison with response rates for established drugs showed the strong efficacy of Sagopilone in non–small cell lung cancer. In gene expression analyses, Sagopilone induced tubulin isoforms in all tumor samples, but genes related to mitotic arrest only in responder models. Moreover, tumors with high expression of genes involved in cell adhesion/angiogenesis as well as of wild-type TP53 were more likely to be resistant to Sagopilone therapy. As suggested by these findings, Sagopilone was combined with Bevacizumab and Sorafenib, drugs targeting vascular endothelial growth factor signaling, in Sagopilone-resistant models and, indeed, antitumor activity could be restored.Conclusion: Analyses provided here show how preclinical studies can provide hypotheses for the identification of patients who more likely will benefit from new drugs as well as a rationale for combination therapies to be tested in clinical trials. Clin Cancer Res; 16(5); 1452–65

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

Objective: Increased numbers of removed lymph nodes (LN) are resulting in more accurate staging of the patient (Will-Rogers-Phenomenon). This study evaluates dependence of lymph node sample size to 1) Will-Rogers-Phenomenom, 2) influence of sample size on overall survival and in terms of 3) morbidity and mortality. Methods: 131 patients after pulmonary resection were retrospectively analysed concerning surgery, number of removed lymph nodes, stage, complications and survival. Patients were stratified according to the median number of lymph nodes in two groups (A

Objective: In chemotherapy for non-small-cell lung cancer (NSCLC), some patients seem to exhibit an intrinsic resistance or develop an acquired resistance under treatment. Results on resistance markers for possible treatment failure as shown in studies on selected lung cancer cell lines could not be completely confirmed in clinical trials. As these conflicting data require further research, we created a model between cell culture and the clinical need to study this problem. Methods: Our study was based on patient-derived NSCLC xenografts in a mouse model, which revealed a high coincidence with the original tumour. Protein and messenger RNA (mRNA) expression of known resistance markers (breast cancer resistance protein (BCRP), multidrug resistance P-glycoprotein (MDR), lung cancer-related protein (LRP) and multidrug resistance protein 1 (MRP1)) were analysed by real-time polymerase chain reaction (PCR) and immunoblotting in 24 xenografts. Chemosensitivity to etoposide, carboplatin, gemcitabine, paclitaxel, cetuximab and erlotinib was determined in in vivo xenograft experiments and compared with the protein and mRNA expression of the multidrug resistance markers. Results: With the exception of a single correlation between chemosensitivity and mRNA expression of etoposide and bcrp (mRNA expression of BCRP), we found no significant correlation between the response rates and protein- and mRNA expression levels in our 24 xenografts. The present results indicate that in vivo expression levels of multidrug resistance proteins and their mRNAs may not play a comparable role in chemoresistance of NSCLC, as pointed out in selected tumour cell lines. Conclusions: Patient-derived xenografts allow detailed investigation of therapy-related markers and their dynamic regulation in a well-standardised and clinically related way. As a consequence of our investigations, we regard multidrug resistance to be a multifactorial phenomenon, in which more factors than the markers analysed by the present study may be involved.

Objective: Despite new technologies, mediastinoscopy remains the gold standard for mediastinal staging of lung cancer even though the procedure is not standardised. Introduction of video-mediastinoscopy (VM) may help to overcome this problem as it better visualises the anatomy and allows a more uniform dissection than conventional mediastinoscopy (CM). Does the use of VM result in more lymph node tissue, higher accuracy and lower complication rates as compared to CM? Methods: All mediastinoscopies from June 2003 to December 2005 were analysed. In a protocol surgeons documented location of lymph node stations, number of lymph nodes resected or biopsied and technique (VM or CM). Two groups were created for analysis: group 1 (n = 366) consisting of all mediastinoscopies was reviewed for complication rates; group 2 included all patients with lung cancer who had a pN0 status by mediastinoscopy and underwent subsequent thoracotomy (n = 171). This group was studied for the number of lymph nodes resected or biopsied according to the technique (VM or CM), on accuracy and negative predictive value. Results: Of 366 mediastinoscopies, 132 were CM (36.1 %) and 234 VM (63.9%). Complications occurred in 17 patients(4.6%): 9 recurrent laryngeal nerve palsies (VM 2.1%, CM 3.0%), 5 mediastinal enlargement on routine chest radiography interpreted as postoperative bleeding (VM 0.9%, CM 2.3%), pneumonia (1), intraoperative laceration of the pleura (1) and main bronchus (1), both corrected during the procedure (all VM 1.3%). No intraoperative haemorrhage or death occurred. VM resected more lymph nodes (mean 8.1, range 3-25) then CM (mean 6.0, range 3-11), for all mediastinoscopies the mean lymph node yield was 7.6 (range 3-25). Comparison of lymphadenectomy via thoracotomy in patients classified pN0 by mediastinoscopy (n = 171) showed an accuracy of 87.9% for VM versus 83.8% for CM (85.8% for all mediastinoscopies) with a negative predictive value of 0.83 for VM and 0.81 for CM (0.82 for all mediastinoscopies). Conclusion: This study demonstrates that in comparison with CM, VM routinely yields more lymph nodes with fewer complications with a tendency towards better accuracy and negative predictive value. For these reasons, we believe that VM should replace CM as the method of choice. Furthermore VM would allow standardisation, thereby having an advantage in comparison to the less invasive newer staging techniques. This way mediastinoscopy could remain the gold standard despite its invasiveness.

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

The differential outcomes of clinical studies of the targeted therapies for non-small cell lung cancer (NSCLC) indicate that better stratification of patients is required. This could be achieved with the help of patient-derived xenografts (PDX) of epidermal growth factor receptor (EGFR) wild-type patients resistant to erlotinib treatment. To explore the potential of patient-derived NSCLC xenografts to optimize therapy using 24 well-characterized early-stage NSCLC PDX. Patient tumor tissue was transplanted subcutaneously into nude mice. After engraftment, tumors were expanded and the sensitivity was tested. Gene expression analysis was used to identify differentially expressed genes between erlotinib responder (n = 3) and non-responder (n = 21). Tumor tissue was analyzed with TaqMan PCR, immunohistochemistry and ELISA to examine the response of the models. Gene expression analysis revealed vascular endothelial growth factor A (VEGFA) to be up-regulated in erlotinib non-responder. Because of that, the combination of erlotinib with bevacizumab was evaluated in one erlotinib-sensitive and four erlotinib-resistant PDX. Combination treatment was superior to monotherapy, leading to the highest and significant inhibition of tumor growth in all models investigated. A decline of VEGFA protein and an increase of VEGFA-mRNA were observed after bevacizumab treatment. Bevacizumab treatment resulted in a distinct decrease of blood vessel number. This study showed that with the help of preclinical PDX models, drug combinations for therapy improvement can be identified on a rational basis. It was observed that a dual blockage of EGFR and VEGFA was more effective than a monotherapy for the treatment of NSCLC in selected PDX models. PDX could be employed to optimize the treatment of cancer patients.

Tobacco smoking is the leading cause of lung cancer worldwide. Gene expression in surgically resected and microdissected samples of non-small-cell lung cancers (18 squamous cell carcinomas and nine adenocarcinomas), matched normal bronchial epithelium, and peripheral lung tissue from both smokers (n = 22) and non-smokers (n = 5) was studied using the Affymetrix U133A array. A subset of 15 differentially regulated genes was validated by real-time PCR or immunohistochemistry. Hierarchical cluster analysis clearly distinguished between benign and malignant tissue and between squamous cell carcinomas and adenocarcinomas. The bronchial epithelium and adenocarcinomas could be divided into the two subgroups of smokers and non-smokers. By comparison of the gene expression profiles in the bronchial epithelium of non-smokers, smokers, and matched cancer tissues, it was possible to identify a signature of 23 differentially expressed genes, which might reflect early cigarette smoke-induced and cancer-relevant molecular lesions in the central bronchial epithelium of smokers. Ten of these genes are involved in xenobiotic metabolism and redox stress (eg AKR1B10, AKR1C1, and MT1K). One gene is a tumour suppressor gene (HLF); two genes act as oncogenes (FGFR3 and LMO3); two genes are involved in matrix degradation (MMP12 and PTHLH); three genes are related to cell differentiation (SPRR1B, RTN1, and MUC7); and five genes have not been well characterized to date. By comparison of the tobacco-exposed peripheral alveolar lung tissue of smokers with non-smokers and with adenocarcinomas from smokers, it was possible to identify a signature of 27 other differentially expressed genes. These genes are involved in the metabolism of xenobiotics (eg GPX2 and FMO3) and may represent cigarette smoke-induced, cancer-related molecular targets that may be utilized to identify smokers with increased risk for lung cancer.

The Siemens servo 300 A ventilator has an automode function that allows automated weaning of patients from mechanical ventilation. Spontaneous breathing triggers the ventilator. After two spontaneously triggered breaths, the ventilator automatically changes from mandatory mechanical ventilation to spontaneous ventilation. If spontaneous breathing or triggering does not occur, the Siemens servo 300 A ventilator changes from spontaneous ventilation back to mandatory mechanical ventilation. We compared the effects of automated versus conventional protocol-driven weaning on the time until extubation in patients undergoing coronary artery bypass graft (CABG) surgery. In addition, we studied the effects of the mode of weaning on hemodynamic and physiologic parameters.Twenty consecutive male patients without respiratory disease scheduled for CABG at the University Hospital of Regensburg were entered into the study. Patients were randomized to postoperative ventilation with the Siemens 300 A/automode ventilator (group A, n = 10) or with the Siemens 300 ventilator (group B, n = 10). All patients were weaned from ventilation according to a standardized protocol.On average, patients in group A were younger and had lower pulmonary artery pressure (PAP) and higher cardiac output compared to patients in group B. However, patients in group A had longer ischemic and bypass times compared to patients in group B. Postoperative use of analgesia and sedation were similar in both groups. Time from tracheal intubation until extubation was 2 h shorter in patients assigned to automode ventilation compared to patients assigned to conventional ventilation (mean time group A 7.9 h, group B 10.0 h; p = 0.069). Peak airway pressure was reduced by 2 cm H2O at the beginning of spontaneous ventilation in group A compared to group B. After extubation, cardiac index showed a greater increase in patients assigned to group A compared to those in group B.Automode ventilator weaning trended toward more rapid extubation than did conventional protocol-driven ventilation in conjunction with a standardized weaning protocol. Physiologic and hemodynamic factors were better in patients using automode ventilation compared to patients using conventional ventilation. Automode ventilation was well tolerated and did not induce significant adverse effects.

Objectives The therapeutic scheme for non-small cell lung cancer (NSCLC) patients can be improved if adapted to the individual response. For example, 60–70% of adenocarcinoma patients show response to EGFR-tyrosine kinase inhibitors in the presence of mutated EGFR. We searched for additional target molecules involved in the action of the EGFR-tyrosine kinase inhibitor erlotinib in the absence of EGFR mutations, which might be suitable for combinatorial therapy approaches. Materials and Methods Erlotinib-response associated proteins were investigated in patient-derived NSCLC mouse xenografts by reverse-phase protein array technology (RPPA) and Western blotting. A combinatorial treatment approach was carried out in NSCLC cell lines and H1299 mouse xenografts, and subsequently analyzed for consequences in cell growth and signal transduction. Results AMP-activated protein kinase (AMPK) expression was increased in erlotinib responders before and after treatment. In a combinatorial approach, activation of AMPK by A-769662 and erlotinib treatment showed a synergistic effect in cell growth reduction and apoptosis activation in H1299 cells compared to the single drugs. AMPK pathway analyses revealed an effective inhibition of mTOR signaling by drug combination. In H1299 xenografts, the tumor size was significantly decreased after combinatorial treatment. Conclusion Our results suggest that AMPK activation status affects response to erlotinib in distinct lung tumor models.

Bronchial stump insufficiency after pneumonectomy is a severe problem and there is still debate about the appropriate method (transthoracic or transsternal) for reclosure. Access through a sterile operative field for a successful redo-procedure seems to be important so an alternative to the open methods could be the video-mediastinoscopy as it allows approaching the bronchial stump via the mediastinum. Previously in 1996 Azorin performed the first mediastinoscopic reclosure by stapling an early insufficiency after left pneumonectomy. We report the first case to our knowledge of resection and reclosure in bronchial stump insufficiency via mediastinoscopy. An HIV-positive man presented with late bronchial stump insufficiency after left pneumonectomy for lung cancer. The cause was a long bronchial stump and there was no sign of tumour recurrence. Decision was made for a video-mediastinoscopy and resection and reclosure successfully performed by using an endostapler device. Postoperative bronchoscopy at six months revealed a well-healed stump and two years postoperatively the patient is doing well. The mediastinoscopic approach is a novel option in highly selected patients. It warrants minimal surgical trauma; however, one has to be prepared to convert to an open technique immediately.

Tropheryma whipplei is known as the bacterium that causes Whipple's disease, a rare systemic illness typically affecting gastrointestinal tract, joints, and central nervous system. In addition, T whipplei infection may present as an isolated endocarditis. Although previously regarded as a rare condition, T whipplei has been recognized as a major cause of culture-negative endocarditis when integrating specific molecular analysis of surgical material into the diagnostic algorithm. Here, we report the case of a 67-year-old man undergoing mitral valve replacement due to T whipplei endocarditis, and discuss diagnostic and therapeutic implications.

OBJECTIVES The management of chest tubes is one of the most critical aspects in patient care in thoracic surgery, and no consensus exists regarding the ideal chest tube management strategy. METHODS Chest tube management protocols and their effects on chest tube therapy were compared at four German specialist thoracic surgery units. Altogether, 79 patients were stratified for underlying disease and type of surgery. A digital chest drainage system was applied to objectify the presence of air leakages. RESULTS In our analysis, the average length of drainage therapy was 4.9 ± 2.8 days. Different chest tube management protocols resulted in a significant degree of scatter between units (P = 0.0348). Higher arbitrary postoperative suction levels (4 kPa) resulted in earlier chest tube removal than lower suction levels (2 kPa) (4.2 ± 2.4 vs 5.4 ± 3.0 days, P = 0.06). Patient discharge following chest tube removal was delayed on average by 3.2 ± 2.9 days. This delay was not correlated with the previous duration of chest tube therapy (Spearman's ρ=-0.15, P = 0.25) in contrast to the total length of hospital stay (ρ = 0.59, P < 0.001).

For resection of lung metastases computed tomography (CT) is needed to determine the operative strategy. A computer-aided detection (CAD) system, a software tool for automated detection of lung nodules, analyses the CT scans in addition to the radiologists and clearly marks lesions. The aim of this feasibility study was to evaluate the reliability of CAD in detecting lung metastases. Preoperative CT scans of 18 patients, who underwent surgery for suspected lung metastases, were analysed with CAD (September-December 2009). During surgery all suspected lesions were traced and resected. Histological examination was performed and results compared to radiologically suspicious nodes. Radiological analysis assisted by CAD detected 64 nodules (mean 3.6, range 1-7). During surgery 91 nodules (mean 5.0, range 1-11) were resected, resulting in 27 additionally palpated nodules. Histologically all these additional nodules were benign. In contrast, all 30 nodules shown to be metastases by histological studies were correctly described by CAD. The CAD system is a sensible and useful tool for finding pulmonary lesions. It detects more and smaller lesions than conventional radiological analysis. In this feasibility study we were able to show a greater reliability of the CAD analysis. A further and prospective study to confirm these data is ongoing.

Purpose: Characterization of new anticancer drugs in a few xenograft models derived from established human cancer cell lines frequently results in the discrepancy between preclinical and clinical results. To take the heterogeneity of tumors into consideration more thoroughly, we describe here a preclinical approach that may allow a more rational clinical development of new anticancer drugs. Experimental Design: We tested Sagopilone, an optimized fully synthetic epothilone, in 22 well-characterized patient-derived non–small cell lung cancer models and correlated results with mutational and genome-wide gene expression analysis. Results: Response analysis according to clinical trial criteria revealed that Sagopilone induced overall responses in 64% of the xenograft models (14 of 22), with 3 models showing stable disease and 11 models showing partial response. A comparison with response rates for established drugs showed the strong efficacy of Sagopilone in non–small cell lung cancer. In gene expression analyses, Sagopilone induced tubulin isoforms in all tumor samples, but genes related to mitotic arrest only in responder models. Moreover, tumors with high expression of genes involved in cell adhesion/angiogenesis as well as of wild-type TP53 were more likely to be resistant to Sagopilone therapy. As suggested by these findings, Sagopilone was combined with Bevacizumab and Sorafenib, drugs targeting vascular endothelial growth factor signaling, in Sagopilone-resistant models and, indeed, antitumor activity could be restored. Conclusion: Analyses provided here show how preclinical studies can provide hypotheses for the identification of patients who more likely will benefit from new drugs as well as a rationale for combination therapies to be tested in clinical trials. Clin Cancer Res; 16(5); 1452–65

Tumor cells that are nonsensitive to anticancer drugs frequently have a multidrug resistant (MDR) phenotype. Many studies with cell lines and patient material have been done to investigate the impact of different resistance markers at protein and mRNA level in drug resistance but with contradictory outcome. In the present study, 26 well-characterised patient-derived non-small cell lung cancer xenografts were used. The known chemosensitivity to etoposide, carboplatin, gemcitabine, paclitaxel and erlotinib was compared to the protein and mRNA expression of BCRP, LRP, MDR1, and MRP1. Further, four of these xenografts were short-term treated to analyse possible regulation mechanisms after therapeutic interventions. We found a borderline correlation between thebcrpmRNA expression and the response of xenografts to etoposide. All other constitutive mRNA and protein expression levels were not correlated to any drug response and were not significantly influenced by a short term treatment. The present results indicate that the expression levels of MDR proteins and mRNA investigated do not play an important role in the chemoresistance of NSCLC in the in vivo situation.

Tumor cells that are nonsensitive to anticancer drugs frequently have a multidrug resistant (MDR) phenotype. Many studies with cell lines and patient material have been done to investigate the impact of different resistance markers at protein and mRNA level in drug resistance but with contradictory outcome. In the present study, 26 well-characterised patient-derived non-small cell lung cancer xenografts were used. The known chemosensitivity to etoposide, carboplatin, gemcitabine, paclitaxel and erlotinib was compared to the protein and mRNA expression of BCRP, LRP, MDR1, and MRP1. Further, four of these xenografts were short-term treated to analyse possible regulation mechanisms after therapeutic interventions. We found a borderline correlation between the bcrp mRNA expression and the response of xenografts to etoposide. All other constitutive mRNA and protein expression levels were not correlated to any drug response and were not significantly influenced by a short term treatment. The present results indicate that the expression levels of MDR proteins and mRNA investigated do not play an important role in the chemoresistance of NSCLC in the in vivo situation.

Purpose: It was the aim of our study to establish an extensive panel of non-small cell lung cancer (NSCLC) xenograft models useful for the testing of novel compounds and for the identification of biomarkers.Experimental Design: Starting from 102 surgical NSCLC specimens, which were obtained from primarily diagnosed patients with early-stage tumors (T2/T3), 25 transplantable xenografts were established and used for further investigations.Results: Early passages of the NSCLC xenografts revealed a high degree of similarity with the original clinical tumor sample with regard to histology, immunohistochemistry, as well as mutation status. The chemotherapeutic responsiveness of the xenografts resembled the clinical situation in NSCLC with tumor shrinkage obtained with paclitaxel (4 of 25), gemcitabine (3 of 25), and carboplatin (3 of 25) and lower effectiveness of etoposide (1 of 25) and vinorelbine (0 of 11). Twelve of 25 NSCLC xenografts were >50% growth inhibited by the anti-epidermal growth factor receptor (EGFR) antibody cetuximab and 6 of 25 by the EGFR tyrosine kinase inhibitor erlotinib. The response to the anti-EGFR therapies did not correlate with mutations in the EGFR or p53, but there was a correlation of K-ras mutations and erlotinib resistance. Protein analysis revealed a heterogeneous pattern of expression. After treatment with cetuximab, we observed a down-regulation of EGFR in 2 of 6 sensitive xenograft models investigated but never in resistant models.Conclusion: An extensive panel of patient-derived NSCLC xenografts has been established. It provides appropriate models for testing marketed as well as novel drug candidates. Additional expression studies allow the identification of stratification biomarkers for targeted therapies.

Comprehensive expression analysis using microarrays has identified a number of differentially expressed genes in smoke-exposed bronchial epithelium and non-small cell lung cancers (NSCLCs). To evaluate the prognostic relevance of these proteins in NSCLCs, we used immunohistochemistry to investigate the expression of beta-catenin (CTNNB1), dickkopf, Xenopus, homolog of 3 (DKK3 gene), fibroblast growth factor receptor 3 (FGFR3), fragile histidine triad (FHIT), tumor protein p53 (TP53), mucin1 (MUC1), topoisomerase II alpha (TOP2A), and glutathione S-transferase-Pi (GST) in a cohort of patients (n = 125). We correlated the expression data with clinicopathologic features and clinical outcome. In addition, SNaPshot multiplex assays (Applied Biosystems, Darmstadt, Germany) and restriction fragment length polymorphism analysis were used to screen for activating point mutations at the hot spots of FGFR3 in a cohort of 30 samples of NSCLC. Using Kaplan-Meier analysis, we observed significantly better overall survival in adenocarcinomas compared with squamous cell cancers (P = .049). Loss of FHIT expression showed a strong association with shorter overall survival in both histologic types of NSCLC (squamous cell cancers, P < .001; adenocarcinomas, P = .001). In adenocarcinomas, the cytoplasmic expression of beta-catenin was associated with shorter survival (P = .012); MUC1 expression was associated with worse prognosis in patients with squamous cell cancers (P = .002). The nuclear staining of TP53 (P = .008) and TOP2A (P = .059) was associated with cancers without lymphonodal metastases. A correlation with positive staining of TOP2A (P = .03) and FGFR3 positivity (P = .057) was found in adenocarcinomas of male patients. Positive MUC1 stainings were associated with squamous cell cancers of male patients (P = .03). DKK3 expression did not show any significant association with clinical outcome or pathologic features. The screening of the FGFR3 sequence in lung cancers showed only wild-type sequences and did not detect mutations in the known hot spots for FGFR3 mutations. We conclude that the immunohistochemical loss of FHIT expression and the positivity for beta-catenin and MUC1 in NSCLC are useful prognostic markers, whereas the variable expression of TP53, TOP2A, and FGFR3 in relation to the different histologic types of NSCLC and sex of the patients is suggestive for different underlying molecular pathways.

Adult respiratory distress syndrome attributable to viral pneumonia is described mainly in immunodeficient persons. Adult respiratory distress syndrome caused by human herpes virus 6 is extremely rare, with only 1 case reported in the literature. The following is the case of a young woman who developed fatal pulmonary failure most probably attributable to human herpes virus 6 pneumonia.

Most lung cancer is attributed to long-term smoking. In order to define chromosomal regions with an accumulation of smoking-related early molecular damage, we applied 15 microsatellite markers at 8 chromosomal regions (2q35-q36, 3p21.3, 3p14.2, 3p25, 10q11.2, 11p14-15, 12p13.1-p12.3 and 12q14) in an allelotyping study. We studied samples of 42 patients with primary non-small cell lung cancer (NSCLC) (25 squamous cell carcinomas, 13 adenocarcinomas, 2 large cell and 2 bronchioalveolar carcinomas) to compare the frequency of allelic loss in cancer tissue of smokers with matched bronchial epithelium. As a control group we used 11 samples of non-smokers. In NSCLC we found significantly higher frequencies of loss of heterozygosity (LOH) than in matched tumor free bronchial epithelium (p = 0.007). Most frequently, allelic loss was detected in NSCLC at chromosome 3p [3p25 (46%), 3p21.3 (45%), 3p14 (40%)], at 2q35 (24%), 12p12 (29%) and 12q14 (13%), but infrequently at 10q11 (7%) and 11p14-15 (5%). In corresponding histological normal bronchial epithelium, the highest percentage of LOH was found at chromosome 3p [3p21 (17%), 3p25 (12%), 3p14 (9%)] and chromosome 2q (2q35-q36) (17%) and 12p (12p12-p13) (12%). LOH in histologically normal bronchial epithelium was significantly associated with long-term smoking (p = 0.048), especially at chromosome 12p12 (p = 0.018). Our results demonstrate two further deletion hot spots at the chromosomal region 2q35-q36 and 12p12-p13 in tumor tissue of NSCLC and matched histological normal bronchial epithelium of long-term smokers, reflecting a phenomenon referred to as 'field cancerization'. These chromosomal regions represent interesting loci for potential NSCLC associated tumor suppressor genes and could be useful as screening markers for molecular risk assessment of smokers.

Genetic alterations at chromosome arm 8p are associated with advanced disease and poor patient outcome in several types of malignant tumors. We studied the frequency of microsatellite instability (MSI) and loss of heterozygosity (LOH) at chromosome 8p in early stage non-small cell lung cancer (NSCLC) of 47 patients with stage I or II disease (25 squamous cell carcinomas and 22 adenocarcinomas). Microsatellite analysis was performed after laser microdissection using 5 polymorphic tetranucleotide microsatellite markers and 4 dinucleotide markers at chromosome 8p. A pentanucleotide repeat marker at the chromosomal locus 17p13.1 (TP53.Alu) was also analyzed. Expression of the mismatch repair (MMR) proteins hMSH2, hMSH6 and hMLH1 was evaluated by immunohistochemistry. Microsatellite instability (MSI) in at least 2 markers was detected in 9 of 47 patients (19.1%) and was predominantly found at tetranucleotide repeats. Sixteen of 47 (34.0%) NSCLC demonstrated LOH at chromosome 8p. All MSI-positive tumors showed normal expression of the MMR proteins. The presence of MSI at chromosome 8p was associated with lymph node metastasis (p=0.02), squamous differentiation (8/25; 32%-p=0.03), and the presence of LOH at the p53 locus (p=0.06). None of the other investigated clinical, pathologic or molecular factors correlated with MSI. Our study showed that an elevated MSI at selected tetranucleotide sequences (EMAST) on chromosome 8p is frequent in early stage squamous cell carcinomas of the lung with lymphatic spread. The tetranucleotide marker panel used in this study was able to indicate lymph node metastasis and high risk disease in patients with resectable squamous cell lung cancer.

Cerebral phaeohyphomycosis caused by Cladophialophora bantiana, a dematiaceous fungus, is a rare disease. The majority of cases have been reported among immunocompetent patients; only 4 cases have been published that describe transplantation patients. The overall prognosis is poor. Surgical therapy in combination with chemotherapy with itraconazole is recommended. We report the case of a heart transplant recipient with cutaneous, cerebral, and lung manifestation of Cladophialophora bantiana who died despite surgical and systemic, high-dosage itraconazole treatment.

Objective: To investigate the effect of short atrioventricular (AV) delay dual-chamber pacing on mean arterial pressure (MAP) and stroke volume index (SVI) in patients with poor left ventricular (LV) function after cardiac surgery. Design: A prospective study. Setting: A university hospital, single-center study. Participants: The study group consisted of 20 patients aged 63 ± 9 years with a left ventricular ejection fraction (LVEF) less than 30%. The control group consisted of 20 patients aged 61 ± 10 years, with an LVEF greater than 50%. Interventions: Immediately after routine coronary artery bypass grafting (CABG) the AV delay was shortened from 160 to 40 milliseconds in atrial-paced (DDD) mode and from 100 to 40 milliseconds in atrial-sensed ventricular stimulation (VDD) mode. MAP was on-line monitored and SVI was calculated by thermodilution. In one patient with an LVEF of 18% (case study), transmitral flow velocity and LV isovolumetric relaxation time were assessed using Doppler echocardiography during VDD pacing at 40-, 80-, and 120-millisecond AV delay. Results: Short-AV delay DDD pacing decreased MAP in the control group (84.3 ± 9 v 75.7 ± 9 mmHg; p < 0.05) and SVI in both groups (study group, 35.9 ± 7 v 31.7 ± 7 mL/m2; control group, 35.3 ± 6 v 31.0 ± 6 mL/m2; p < 0.05). Shortening the AV delay had no influence on MAP and SVI during VDD pacing. During the echocardiographic case study, AV delay shortening distinctly modified ventricular filling patterns. Optimal LV filling and transmitral flow were achieved with an intermediate AV delay of 80 milliseconds. Conclusion: Dual-chamber pacing with nonphysiologic short AV delay failed to improve acute hemodynamics in patients with poor LV function after CABG. Short AV delay VDD pacing was superior to DDD pacing in both normal and impaired LV function. The use of Doppler echocardiography enabled optimization of the AV delay on the basis of LV filling patterns.

Objective: For patients with atrioventricular block single lead atrial synchronous ventricular pacing (VDD) may have advantages compared to conventional dual chamber pacing (DDD) since it eliminates the need for an atrial lead. The purpose of this study was to investigate the clinical performance of a novel VDD pacemaker and the reliability of atrial sensing via the ‘floating’ atrial electrode. Methods: 31 patients (10 females; age 649 13 years) underwent an implantation of a VDD pacemaker system (Intermedics UNITY). The patients were analyzed with regard to implantation parameters, complications and postoperative atrial sensing performance using the diagnostic data of the pacemaker memory. The mean follow-up was 6.3 (1‐18) months. Results: The implantation procedure did not differ from that of conventional single chamber pacemakers. Dislocation of a ventricular electrode was the only complication observed. The P wave at implantation was 1.69 0.9 mV and dropped to 0.990.4 mV at predischarge. During follow-up the atrial sensing threshold remained stable. The atrial sensing performance (percentage of atrial synchronous ventricular complexes) after reprogramming the highest atrial sensitivity was 99.7%. Two patients (6%) developed atrial fibrillation. 29 patients (94%) remained in VDD mode as primarily intended. Conclusions: From these results it is concluded that VDD pacing represents an excellent alternative in patients with atrioventricular block and intact sinus node function. The atrial sensing was found to be reliable. © 1997 Elsevier Science B.V.

A total of 164 patients with a mean age of 78.6±2.7 years and often critical preoperative conditions [New York Heart Association (NYHA) class III + IV, 78.7%] underwent cardiac operations [coronary artery bypass grafting (CABG) 97; valve replacement, 33; CABG + valve replacement, 18; replacement of ascending aorta and others, 18] with an in-hospital mortality of 8.5% (n=14). Follow-up was completed for 147 patients (98%) after 16.5±10.8 months. Most patients were in a good clinical condition (NYHA I + II, 79.6%; late deaths, 8 patients, 5.4%) and more than 95% of patients lived at home or with relatives. Charges for hospital treatment dropped to one third after the hospitalization period for surgery in the first postoperative year compared to the year immediately preceding the cardiac operation.

We have recently described an extended panel of new primary human CRC xenografts and their preclinical chemo-sensitivity to anti-cancer drugs (Poster 1584/3 AACR Annual Meeting 2010). These models consider more thoroughly the heterogeneity and genetic instability of tumors in a way ultimately enabling treatment decisions on an individualized basis. The approach is based on large scale screening for activity in new in vivo models combined with extensive characterization of these models (gene expression and mutations). Bioinformatic methods are used to further evaluate the mechanism of action of new drugs in colorectal cancer, to investigate possible resistance mechanisms and provide a rationale for combination therapies. Here we describe the evaluation of an antiangiogenic treatment in this panel of primary patient derived colorectal cancer models. Primary colon carcinoma tissue samples were collected using a standardized procedure. Tumor pieces were transplanted into immunodeficient mice immediately after surgery. A panel of 28 stably passagable colon cancer xenografts could be established as permanent tumor models. The results obtained so far, show that these patient-derived colon cancer models feature a high coincidence with the original tumor regarding histology and gene expression profiling. Response analysis in a subset of 19 xenografts evaluating the treated-to-control (T/C) ratios of tumor volumes revealed that the human specific VEGF antibody bevacizumab induces a meaningful biological effect (T/C below 50%) in 13 of 19 models. A first comparative genome-wide gene expression analyses of this subset revealed reasonable genes and pathways possibly involved in bevacizumab sensitivity. We will extend this investigation to all 28 tumor models in order to enhance the statistical robustness of the preliminary data. Our approach demonstrates that analysis of drug sensitivity together with bioinformatics correlations can provide candidate markers for further studies. An early implementation of such an approach in the drug development process could be helpful for the identification of predictive markers helping to select tumors likely to benefit from the drug as well as provide a rationale for combinations to be tested in clinical trials. These new strategies may help to better integrate research results in clinical development and may increase the success of new anti-cancer drugs in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4524. doi:1538-7445.AM2012-4524

Almost in parallel with the new millennium processes in drug development for cancer therapy have changed substantially. Insight in the cancer biology have revealed this disease as a complex genetic disorder leading to a high variety of phenotypes among the different individuals. Many tumors present a very specific pattern of molecular changes and this leads to a heterogenous individual response to drugs. The correct identification of predictive biomarkers selecting the most appropriate therapies and avoiding unnecessary treatments for an individual patient is still a challenge. Patient-derived tumor xenografts allow preclinical investigations in a clinically relevant way. We performed investigations to improve the understanding of cancer complexity and to draw rational conclusions for therapy decisions. Tumor models were established by direct transplantation of surgical specimens to immunodeficient mice and were maintained in early passages. We have shown a high correlation between original patient sample and xenograft both at gene and protein level. We established several large panels of tumor models: i.e. 10 breast, 30 colorectal, 25 lung, 6 ovarian, 10 sarcomas, and 30 leukemias (25 ALL and 5 AML). During the characterization of the xenografts for response towards clinically used anticancer drugs, the analysis for mutations and of gene expression revealed interesting correlations. In NSCLC models upregulation of hypoxia marker genes correlated with resistance to microtubule targeted drugs. In the colon cancer xenografts KRAS mutations predicted resistance to cetuximab. We further identified a potential set of 20 genes which may be predictive for oxaliplatin response. These results demonstrate, that the ability to assess anti-tumor activity in well-characterized xenografts in correlation with particular genetic or molecular characteristics may support the development of new therapeutic regimens. Conclusions from what we discussed are:drug discovery, systems biology, and translational research are moving together to address all the new hallmarks of cancer - increasing the success rate of drug discoverypanels of patient derived xenograft models represent an important tool for translational research to address clinically relevant questions in a standardized and strictly controlled fashionpredictive value of the preclinical models is increasing steadily - they show a high concordance with the clinical specimens concerning marker expression and response to therapy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A14.

Improved methods for the preclinical characterization of anti-cancer drugs in larger panels of primary lung cancer xenograft models have recently described (Fichtner et al. Clin Cancer Res. 2008;14(20):6456-68). These models consider more thoroughly the heterogeneity and genetic instability of tumors in a way ultimately enabeling treatment decisions on an individualized basis. The approach is based on large scale screening for activity in new in vivo models combined with extensive characterization of these models (gene expression and mutations). Bioinformatic methods are then used to further evaluate the mechanism of action of new drugs, to investigate possible resistance mechanisms and provide a rationale for combination therapies. Here we describe the development of a panel of primary patient derived colorectal cancer models. Primary colon carcinoma tissue samples were collected using a standardised procedure. Tumour pieces were transplanted onto immunodeficient mice immediately after surgery. A panel of 28 stably passagable colon cancer xenografts could be established as permanent tumour models. The results obtained so far, show that these patient-derived colon cancer models feature a high coincidence with the original tumour regarding histology and gene expression profiling. Response analysis according to clinical trial criteria revealed that the standard drug Oxaliplatin induce a overall response in 8 of the 28 xenograft models. Gene expression analyses were performed to identify predictive profiles for Oxaliplatin sensitivity. As one example, the effectiveness of Oxaliplatin seems to be related to the differential expression of genes which products are involved in the wnt pathway. The analysis of drug sensitivity in this set of models together with bioinformatics coorelations will provide hypotheses for further studies to identify patients that will more likely benefit from a drug as well as provide a rationale for combinations to be tested in clinical trials. These new approaches may help to better integrate research results in clinical development and may increase the success of new anti-cancer drugs in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1584. doi:10.1158/1538-7445.AM2011-1584