Volker Steger

Generation and transplantation of an autologous vascularized bioartificial human tissue.

Background. The lack of transplant vascularization forecloses the generation and clinical implementation of bioartificial tissues. We developed techniques to generate a bioartificial human tissue with an innate vascularization. The tissue was implanted clinically as proof of concept to evaluate vascular network thrombogenicity and tissue viability after transplantation. Methods. A porcine small bowl segment was decellularized in a two-step procedure, preserving its vascular structures. The extracellular matrix was characterized quantitatively for DNA residues and protein composition. The vascular remainings were reseeded with human endothelial cells in a dynamic tissue culture. The engineered tissue was characterized by (1) histology, (2) immune-histology, (3) life-dead assay, and (4) metabolic activity. To evaluate the tissue capabilities, it was implanted clinically and recovered after 1 week. Results. Tissue preparation with sodium desoxycholate monohydrate solution resulted in an incomplete decellularization. Cell residues were removed by additional tissue incubation with DNAse. The human endothelial cells formed a viable endothelium inside the primarily porcine extracellular matrix, expressing CD31, Flk-1, and vascular endothelium-cadherin. The metabolic activity of the bioartificial tissue increased continuously over time in vitro. Clinical tissue transplantation confirmed vessel patency and tissue viability for 1 week. Conclusions. The feasibility to bioengineer a human tissue with an innate vascularization has been shown in vitro and the clinical setting. These results may open the door for the clinical application of various sophisticated bioartificial tissue substitutes and organ replacements.

Phase II Trial of a Trimodality Regimen for Stage III Non-Small-Cell Lung Cancer Using Chemotherapy As Induction Treatment With Concurrent Hyperfractionated Chemoradiation With Carboplatin and Paclitaxel Followed by Subsequent Resection: A Single-Center Study

PURPOSE We started a phase II trial of induction chemotherapy and concurrent hyperfractionated chemoradiotherapy followed by either surgery or boost chemoradiotherapy in patients with advanced, stage III disease. The purpose is to achieve better survival in the surgery group with minimum morbidity and mortality. PATIENTS AND METHODS Patients treated from 1998 to 2002 with neoadjuvant chemoradiotherapy and surgical resection for stage III NSCLC were analyzed. The treatment consisted of four cycles of induction chemotherapy with carboplatin/paclitaxel followed by chemoradiotherapy with a reduced dose of carboplatin/paclitaxel and accelerated hyperfractionated radiotherapy with 1.5 Gy twice daily up to 45 Gy. After restaging, operable patients underwent thoracotomy. Inoperable patients received chemoradiotherapy up to 63 Gy. Study end points included resectability, pathologic response, and survival. Results One hundred twenty patients were enrolled; 25% patients had stage IIIA, 73% had stage IIIB, and 2% stage IV. After treatment, 47.5% had downstaging, 29.2% had stable disease, and 23.3% had progressive disease. Thirty patients (25%) were not eligible for operation because of progressive disease, stable disease, and/or functional deterioration with one treatment-related death. The 30-day mortality was 5% in patients who underwent operation. The 5-year survival rate for 120 patients was 21.7%, and it was 43.1% in patients with complete resection. In postoperative patients with stage N0 disease, 5-year survival was 53.3%; if stage N2 or N3 disease was still present, 5-year survival was 33.3%. CONCLUSION Staging and treatment with chemoradiotherapy and complete resection performed in experienced centers achieve acceptable morbidity and mortality.

Clinical tracheal replacement: transplantation, bioprostheses and artificial grafts

The replacement of significant lengths of tracheal tissue still remains the unrivalled ‘holy grail’ of thoracic surgery. As a result of continuous developments and improvements in tracheal surgery throughout the last five decades, most tracheal lesions can now be resected and primary reconstruction effected safely. Today, very few patients present extensive tracheal lesions that necessitate tracheal replacement. The spectrum of tracheal substitutes ranges from autologous tissue flaps and patches to synthetic stents and prostheses to tissue-engineered scaffolding. In this review, the clinical achievements and conceivable developments of applying human allografts and biological and artificial prostheses will be discussed.

Trimodal Therapy for Histologically Proven N2/3 Non–Small Cell Lung Cancer: Mid-Term Results and Indicators for Survival

BACKGROUND Surgery alone for stage III non-small cell lung cancer provides a 5-year survival of 20% and competes with multimodal treatments. In 1999, a trimodal protocol was implemented at the Schillerhöhe Clinic. The aim of this study was to verify the feasibility and outcome of this trimodal protocol including survival, risk factors for survival, and comorbidity in a single institution. METHODS Included were all patients with potentially resectable, previously untreated stage III non-small cell lung cancer operated on between February 1999 and May 2006 in the General Thoracic Surgery Unit of the Schillerhöhe Clinic following the same neoadjuvant protocol. Treatment-related morbidity, recurrence, survival after R0 resection, and risk factors for survival (pN0 after trimodal therapy, downstaging of International Union Against Cancer stage, T downstaging, N downstaging, regression rate, and histologic type of tumor) were analyzed. RESULTS From 107 patients with stage III non-small cell lung cancer, 55 patients with mediastinoscopy-positive N2 or N3 were eligible for this study. Forty patients (72%) had the effect of International Union Against Cancer downstaging. Treatment-related comorbidity was 54% with hospital and 120-day mortality of 3.6% and 5.4%, respectively. Overall mean survival (Kaplan-Meier) was 43 months (95% confidence interval, 35 to 52) with an estimated 5-year survival rate of 49%. In multivariate testing, International Union Against Cancer downstaging after trimodal therapy achieved a level of significance (p = 0.031), and patients with UICC-downstaging after trimodal therapy had a mean survival of 53 months (95% confidence interval, 44 to 63) with an estimated 5-year survival rate of 60%. CONCLUSIONS Neoadjuvant trimodal treatment for histologically proven N2 or N3 stage III non-small cell lung cancer is promising and can, like no other approach at present time, considerably improve 5-year survival rates up to 63% in selected patients.

Long-term efficiency of endoscopic thoracic sympathicotomy: survey 10 years after surgery

Immediate and short-term results of endoscopic thoracic sympathectomy (ETS) for primary hyperhidrosis are good. Adverse effects have been identified clearly and are supposed to decrease with time. In this institutional report, the long-term results of ETS with regard to efficacy, side effects and patient satisfaction are presented. Fifteen patients were included and mean follow-up time was 12+/-2 years. ETS success rate, rate of compensatory sweating and degree of patient satisfaction were assessed. We detected 8 patients (53%) complaining about a decent to moderate recurrence of hand sweating and compensatory and gustatory sweating were observed in 9 (60%) and 5 (33%) patients, respectively. Reported side effects related to surgery were paresthesias of the upper limb and the thoracic wall in 8 patients (53%) and recurrent pain in the axillary region in one. At an average 12 years after surgery, 47% of patients were satisfied with the treatment results, 40% were disappointed. Six patients (40%) affirmed they would ask for the operation if it were to be redone. Our findings indicate that results of ETS deteriorate and compensatory sweating does not improve with time. It is mandatory to inform patients of the potential long-term adverse effects before surgery.

Pneumonectomy: calculable or non-tolerable risk factor in trimodal therapy for Stage III non-small-cell lung cancer?

OBJECTIVES Lung cancer is the leading cause of death in cancer statistics throughout developed countries. While single surgical approach provides best results in early stages, multimodality approaches have been employed in advanced disease and demonstrated superior results in selected patients. With either full-dose chemotherapy and/or radiotherapy, patients usually have a poor general condition when entering surgical therapy and therefore neoadjuvant therapy can lead to a higher morbidity and mortality. Especially in the case of pneumonectomy as the completing procedure, mortality rate can exceed over 40%. Therefore, chest physicians often shy away from recommending pneumonectomy as final step in trimodal protocols. We analysed our experience with pneumonectomy after neoadjuvant chemoradiotherapy in advanced non-small-cell lung cancer (NSCLC) with a focus on feasibility, outcome and survival. METHODS Retrospective, single-centre study of 146 patients with trimodal neoadjuvant therapy for NSCLC Stage III over 17 years time span. Follow-up was taken from our own outpatient files and with survival check of central registry office in Baden-Württemberg, Germany. RESULTS A total of 118 men and 28 women received 62 lobectomies, 6 bi-lobectomies and 78 pneumonectomies after two different neoadjuvant protocols for Stage III NSCLC. Overall morbidity rate was 53 and 56% after pneumonectomy. Overall hospital mortality rate was 4.8 and 6.4% after pneumonectomy. Overall median survival rate was 31 months with a 5-year survival rate of 38% (Kaplan-Meier). Pneumonectomy, right-sited pneumonectomy and initial T- and N-stages were no risk factors for survival (log-rank test). Significant factors for survival were ypT-stage, ypN-stage, yUICC-stage in univariate testing (log-rank test) and ypUICC-stage in multivariate testing (Coxs regression). CONCLUSIONS Pneumonectomy in neoadjuvant trimodal approach for Stage III NSCLC can be done safe with acceptable mortality rate. Patients should not withhold from operation because of necessitating pneumonectomy. Not the procedure but the selection, response rate and R0-resection are crucial for survival after trimodal therapy in experienced centres.

A new strategy in the treatment of chemoresistant lung adenocarcinoma via specific siRNA transfection of SRF, E2F1, Survivin, HIF and STAT3 †

OBJECTIVES According to the actual treatment strategies of lung cancer, the current therapeutic regimen is an individualized, multidisciplinary concept. The development of chemoresistance in the last decade represents the most important obstacle to an effective treatment. In our study, we examined a new therapeutic alternative in the treatment of multiresistant lung adenocarcinoma via siRNA-specific transfection of six crucial molecules involved in lung carcinogenesis [serum response factor(SFR), E2F1, Survivin, hypoxia inducible factor1 (HIF1), HIF2 and signal transducer and activator of transcription (STAT3)]. METHODS Three chemoresistant A549 adenocarcinoma cells were cultured under standard conditions at 37°C and 5% CO2. The chemoresistance against Vinflunine, Vinorelbine and Methotrexate was induced artificially. The A549 cells were transfected for 2 h at 37°C with specific siRNA targeting SRF, E2F1, Survivin, HIF1, HIF2 and STAT3 in a non-viral manner. The efficiency of siRNA silencing was evaluated via quantitative real-time polymerase chain reaction, whereas the surviving cells after siRNA transfection as predictor factor for tumoural growth were analysed with a CASY cell counter 3 days after transfection. RESULTS The response of the chemotherapeutic resistant adenocarcinoma cells after siRNA transfection was concentration-dependent at both 25 and 100 nM. The CASY analysis showed a very effective suppression of adenocarcinoma cells in Vinorelbine, Vinflunine and Methotrexate groups, with significantly better results in comparison with the control group. CONCLUSIONS In our study, we emphasized that siRNA interference might represent a productive platform for further research in order to investigate whether a new regimen in the treatment of multiresistant non-small-cell lung cancer could be established in vivo in the context of a multimodal cancer therapy.

Small interfering RNA-mediated suppression of serum response factor, E2-promotor binding factor and survivin in non-small cell lung cancer cell lines by non-viral transfection †

OBJECTIVES Serum response factor (SRF), E2F1 and survivin are well-known factors involved in a multitude of cancer-related regulation processes. However, to date, no suitable means has been found to apply their potential in the therapy of non-small cell lung cancer (NSCLC). This study deals with questions of small interfering ribonucleic acid (siRNA) transfection efficiency by a non-viral transfection of NSCLC cell-lines and the power of siRNA to transiently influence cell division by specific silencing. METHODS Different NSCLC cell lines were cultured under standard conditions and transfected, with specific siRNA targeting SRF, E2F1 and survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as controls. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for messenger RNA (mRNA) expression levels. Additionally, transfection efficiency was evaluated by flow cytometry. The analysis of cell proliferation was determined with a CASY cell counter 3 days after transfection with SRF or SCR-siRNA. RESULTS Transfection of the NSCLC cell lines with specific siRNAs against SRF, E2F1 and survivin resulted in a very considerable reduction of the intracellular mRNA concentration. CASY confirmation of cell viability demonstrated an excellent survival of the cell lines treated with non-specific siRNA, in contrast to with application of specific siRNA. CONCLUSIONS This study reports a reliable transfectability of NSCLC-cell lines by siRNA, initially in a non-viral manner, and a reproducible knockdown of the focussed targets, consequently leading to the death of the tumour cells. This constitutes a strong candidate for a new assessment strategy in the therapy of non-small cell lung cancer.

Surgery on unfavourable persistent N2/N3 non-small-cell lung cancer after trimodal therapy: do the results justify the risk?

OBJECTIVES Persistent mediastinal lymph node metastasis after neoadjuvant therapy is a significant negative indicator for survival. Even though there is still no consensus on the matter, some authors advocate a thorough restaging prior to surgery and deny surgery in cases of persistent N2 because of the poor outcome. We analysed our results after trimodal therapy in pN2/N3 stage III non-small-cell lung cancer (NSCLC) and persistent mediastinal lymph node metastasis after neoadjuvant chemoradiotherapy. METHODS We conducted a retrospective cohort analysis of 167 patients who received trimodal therapy for stage III NSCLC. Progression-free interval and survival were calculated. T-stage, N-stage, ypT-stage, ypN2/3-stage and surgical procedure were tested as risk factors. RESULTS Eighty-three patients with potentially resectable initial pN2/3 underwent 44 pneumonectomies and 76% extended resections. Thirty-five patients showed persistent mediastinal lymph node metastasis after trimodal therapy. Treatment-related comorbidity after an operative therapy was 58%. Hospital mortality was 2.4%. The ypT- and ypN2/N3 stages were significant risk factors and, in the case of persistent mediastinal lymph node metastasis, median progression-free period was 17 months and median survival time was 21 months. CONCLUSIONS Persistent but resectable N2/N3 after chemoradiotherapy in stage III NSCLC is the least favourable subgroup of patients in neoadjuvant approaches. If surgery can be carried out with curative intent and low morbidity, completing trimodal therapy is justified, with an acceptable outcome.

Impact of neo-adjuvant radiochemotherapy on bronchial tissue viability

OBJECTIVE In the treatment of advanced stages of lung cancer, increasingly more multimodality approaches applying radiotherapy and/or chemotherapy in a neo-adjuvant setting are being introduced. The impact of induction therapy, especially radiotherapy, on bronchial tissue viability has not been investigated so far. METHODS In 2008, we determined the tissue viability of bronchial segments obtained during surgery in 45 consecutive patients, including patients after neo-adjuvant radiochemotherapy (RCTX). Bronchial tissue viability was analysed by histology, life-dead assay and cell proliferation in tissue-specific culture media. Biomedical findings were compared with the clinical course of the patients. RESULTS Tissue samples of 44 patients were included into this study. Fourteen patients (32%) had undergone neo-adjuvant RCTX. Histology and life-dead assay of the bronchial segments did not show significant differences. While patient age, sex, tumour entity and site of resection had no influence on cell proliferation in vitro, previous RCTX resulted in a 46% decrease of bronchial tissue viability (P=0.01). However, this effect was not reflected by the clinical course of the operated patients. CONCLUSIONS Neo-adjuvant RCTX reduces bronchial tissue viability substantially. However, this impairment does not necessarily translate into an increased rate of postoperative bronchial insufficiencies. Standard histological work-up is not sensitive enough to characterise changes in bronchial tissue viability following RCTX.