Bernward Passlick

Frequency and prognostic significance of isolated tumour cells in bone marrow of patients with non-small-cell lung cancer without overt metastases

BACKGROUND Metastasis is generally looked on as a late event in the natural history of epithelial tumours. However, the poor prognosis of patients with apparently localised lung cancer indicates that micrometastases occur often before diagnosis of the primary tumour. METHODS At primary surgery, disseminated tumour cells were detected immunocytochemically in bone marrow of 139 patients with non-small-cell lung carcinomas without evidence of distant metastases (pT(1-4)pN(1-2)M(0)). Tumour cells in bone-marrow aspirates were detected with monoclonal antibody CK2 against cytokeratin polypeptide 18. Patients were followed up for a median of 39 months (range 14-52) after surgery. 215 patients without epithelial cancer (ie, with benign epithelial tumours, non-epithelial neoplasms, or inflammatory diseases) acted as controls. FINDINGS In 83 of 139 (59.7%) patients cytokeratin-positive cells were detected at frequencies of 1 in 100 000 to 1 in 1 000 000. Even without histopathological involvement of lymph nodes (pN(0)), tumour cells were found in 38 of 70 (54.3%) patients. 1 positive cell was found in each of 6 out of 215 controls. Surgical manipulation during primary tumour resection did not affect the frequency of these cells. In Coxs regression analyses, the presence of such cells was a significant and independent predictor for a later clinical relapse in node-negative patients (p=0.028). INTERPRETATION Early dissemination of isolated tumour cells is a frequent and intrinsic characteristic of non-small-cell lung carcinomas. The finding of these cells may help to decide whether adjuvant systemic therapy is required for the individual patient.

Combined transcriptome and genome analysis of single micrometastatic cells

In human cancer, early systemic spread of tumor cells is recognized as a leading cause of death. Adjuvant therapies are administered to patients after complete resectioning of their primary tumors to eradicate the few residual and latent metastatic cells. These therapeutic regimens, however, are currently designed without direct information about the presence or nature of the latent cells. To address this problem, we developed a PCR-based technique to analyze the transcriptome of individual tumor cells isolated from the bone marrow of cancer patients. From the same cells, genomic aberrations were identified by comparative genomic hybridization. The utility of this approach for understanding the biology of occult disseminated cells and for the identification of new therapeutic targets is demonstrated here by the detection of frequent extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) expression which was verified by immunostaining.

Impact of radical systematic mediastinal lymphadenectomy on tumor staging in lung cancer.

The extent of lymphadenectomy in the treatment of non-small cell lung cancer is still a matter of controversy. While some centers perform mediastinal lymph node sampling with resection of only suspicious lymph nodes, others recommend a radical, systematic mediastinal lymphadenectomy (LA) to improve survival and achieve a better staging. Herein we report on the impact of LA on tumor staging in a controlled, prospective, randomized clinical trial comparing lymph node sampling and LA in a total of 182 patients with operable non-small cell lung cancer. Regardless of the type of lymphadenectomy performed, the percentage of patients with pathologic N1 or N2 (sampling: n = 23, 23.0%; LA: n = 22, 26.8%) disease was very similar in both groups, indicating that systematic radical lymphadenectomy is not an essential prerequisite to determine the N stage of a patient. In contrast, the number of patients detected to have lymph node involvement at multiple levels was significantly increased by LA. In the lymph node sampling group only 4 of 23 patients (17.4%) with N2 disease were found to have more than one lymph node level involved, whereas LA results in the detection of excessive N2 disease in 12 of 21 patients (57.2%; p = 0.007), which was associated with a shorter distant metastases-free (p = 0.021) and overall survival. In conclusion, LA is not essential to determine the N stage of a patient, but results in a more detailed staging of the N2 region, which is of prognostic significance. Therefore, it might be useful to identify patients with a higher risk for tumor relapse.

Detection of disseminated lung cancer cells in lymph nodes: Impact on staging and prognosis

BACKGROUND A major reason for the high incidence of tumor recurrences in patients with apparently resectable non-small cell lung cancer is presumably early tumor cell dissemination, which is clearly underestimated by current staging procedures. METHODS In this prospective study we assessed the frequency and prognostic significance of early lymphatic tumor cell spread to regional lymph nodes staged as tumor free by conventional histopathology by applying an immunohistochemical assay using monoclonal antibody Ber-Ep4. RESULTS Ber-Ep4 positive cells were demonstrated in 27 (21.6%) of 125 patients and in 35 (6.2%) of 565 lymph nodes, respectively. Immunohistochemical analysis resulted in an up-staging in 24 of 27 patients. In patients previously staged as having pN0 disease, tumor cells were detected in 11/70 cases (15.7%). Univariate and multivariate survival analysis showed that the detection of minimal nodal tumor cell dissemination was associated with a reduced disease-free survival (log rank test, p = 0.0001; Cox regression model, p = 0.001). CONCLUSIONS The use of immunohistochemistry enables one to identify many patients with regional tumor cell dissemination at the time of operation. These patients might benefit from an adjuvant therapeutic regimen.

Incidence of chronic pain after minimal-invasive surgery for spontaneous pneumothorax

OBJECTIVE Recently, it has been shown that minimal-invasive surgical procedures like operations for spontaneous pneumothorax result in a reduction of pain in the immediate postoperative course. However, little is known on the influence of minimal-invasive thoracic surgery on long term disability. Therefore, we analyzed the incidence of chronic pain in patients after minimal-invasive operation for primary (PSP) or secondary (SSP) spontaneous pneumothorax. METHODS In the study included were 78 patients (PSP: n=59; SSP: n=19; male: 58, female 20) who had been treated at our institution between 1992 and 1995. The median age was 37 years (range: 17-84). The patients were interviewed by a standardized questionnaire or alternatively by phone or in the outpatient clinic. Complete follow up data were obtained from 60 patients which were further analyzed. RESULTS After a median follow up of 59 months (range 35-79) 41 (68.3%) patients were completely free from any complaints. However 19 (31.7%) patients suffered from chronic pain. Two of them (3.3%) required daily oral pain medication. The incidence of chronic complaints was more frequent in patients with pleurectomy (47.1%) as compared to patients with mechanical pleurodesis only (25.6%; P=0.107). On a visual analog pain scale (ranging from 0 to 100) five (8.3%) patients described a pain intensity <10, 12 (20%) patients between 10 and 20 and two (3.3%) patients >50. In the majority of the patients the pain was located in the area of the trocar incisions. Six (10%) patients had a chronic complaints in the ipsilateral shoulder. CONCLUSIONS The incidence of chronic postoperative complaints after minimal-invasive procedures for spontaneous pneumothorax is relatively high. This has to be considered if minimal-invasive procedures are discussed to be an alternative to simple drainage therapy for the first episode of spontaneous pneumothorax.

Isolated tumor cells in bone marrow predict reduced survival in node-negative non-small cell lung cancer

BACKGROUND It recently became evident that isolated tumor cells undetectable by conventional tumor staging are frequently present in bone marrow of patients with apparently localized non-small cell lung cancer (NSCLC). The clinical relevance of this minimal hematogenous tumor cell dissemination is under vigorous debate. METHODS For tumor cell detection in the bone marrow, we used monoclonal antibody CK2 against the epithelial intermediate filament protein cytokeratin 18. The influence of a positive bone marrow finding on clinical outcome was studied in 139 patients with NSCLC postoperatively staged as pT1-4, pN0-2, M0, and R0 after a median follow-up of 66 months (range 48 to 74 months). RESULTS Cytokeratin-18-positive cells in bone marrow were demonstrated in 83 (59.7%) patients at the time of primary surgery and in 6 of 12 representative patients analyzed twice 3 to 18 months after surgery. In patients without histopathological lymph node metastases (pN0; n = 66), the occurrence of 2 or more tumor cells in bone marrow at primary surgery was a strong and independent predictor for overall survival (p = 0.007) in univariate analysis. The multivariate analysis showed a 2.8 times increased risk for shorter survival in patients with disseminated tumor cells versus patients without such cells. Four of the 6 patients with a positive cytokeratin status after surgery developed a tumor recurrence 11 to 44 months after the operation, while none of the patients with a negative bone marrow at all time intervals showed a tumor relapse. CONCLUSIONS Minimal residual bone marrow involvement is an independent prognostic factor for overall survival in patients with node-negative NSCLC, which may help to identify patients in need of an adjuvant systemic therapy. The postoperative persistence or reappearance of tumor cells in bone marrow indicates that these are not only shed cells but rather represent true micrometastasis.

Prognostic impact of matrix metalloproteinase-9 in operable non-small cell lung cancer.

We assessed the clinical impact of MMP‐9 expression on long‐term survival in patients with operable non‐small cell lung cancer (NSCLC). Primary tumors of 143 consecutive patients with NSCLC resected completely and without overt distant metastases (pT1‐4, pN0‐2, M0, R0) were examined for MMP‐9 expression using immunohistochemistry with a polyclonal, affinity‐purified rabbit antibody that recognizes both latent and active MMP‐9. Immunohistochemical staining of tumor cells was evaluated in comparison to normal bronchiolar epithelium that served as an internal positive control. MMP‐9 expression was categorized into negative, ≤5% tumor cells stained; heterogeneous, >5% and <95% tumor cells stained; and homogeneous, ≥95% tumor cells stained at least as intensively as bronchiolar epithelium. The median follow‐up period was 72 months (range = 12–144 months). Homogeneous expression of MMP‐9 was observed in 26 of 143 patients (18.2%) and did not correlate with clinicopathological parameters. Relapse defined as diagnosis of distant metastasis or local recurrence was observed in 78 of the 130 (60%) patients eligible for clinical follow up analysis. Relapse led to cancer‐related death in all of the 78 patients within the observation period. Kaplan‐Meier analysis showed a significant association between homogeneous MMP‐9 expression and shortened cancer‐related survival (log‐rank p = 0.016). Multivariate regression analysis including pT‐status, pN‐status, tumor histology and tumor grading showed an independence of this prognostic impact of homogeneous MMP‐9 expression (p = 0.045). Thus, immunohistochemical evaluation of MMP‐9 expression may provide a basis for the preselection of patients to be included in trials investigating specific protease inhibitor therapy after surgical resection of NSCLC.

Mediastinal lymphadenectomy in non-small cell lung cancer: effectiveness in patients with or without nodal micrometastases - results of a preliminary study.

OBJECTIVES So far it has not clearly been demonstrated that systematic mediastinal lymphadenectomy improves survival in patients with non-small cell lung cancer. One explanation might be that in some patients an early spread of tumor cells has occurred which might not be curable by surgical means. To test this hypothesis lymph nodes of patients which were treated either by lymph node sampling or systematic lymphadenectomy were screened for micrometastatic spread of tumor cells and the influence of nodal micrometastases on the efficacy of lymphadenectomy was analyzed. METHODS Lymph nodes from patients (n=94) which were included in a randomized trial of lymph node sampling (LS, n=41) versus radical systematic lymphadenectomy (LA, n=53) were screened by immunohistochemistry for disseminated tumor cells using the antibody Ber-Ep4. The median observation time was longer than 5 years and follow-up data were available from all 94 patients. Kaplan-Meier curves were calculated and tested for statistical significance using the log-rank test. RESULTS Standard histopathological analysis revealed no lymph node involvement (pN0) in 61 patients, pN1 disease in 13 patients and pN2 disease in 20 patients without significant differences between LA and LS with respect to T-stage, N-stage or age and sex of the patients. By immunohistochemistry a minimal nodal spread of tumor cells was detected in 21 out of 94 patients (LS, n=10 (24%); LA, n=11 (21%)). Similar to the entire group of patients also in the subset of patients with nodal micrometastases the type of lymphadenectomy did not significantly influence the long-term survival (P=0.27 and P=0.39, respectively). In contrast, in patients with a negative immunohistochemical analysis systematic lymphadenectomy resulted in an improved overall survival (P=0.044). CONCLUSIONS Our data provide some evidence that systematic lymphadenectomy improves survival in patients without an early locoregional spread of cancer cells. As long as these patients can not be identified preoperatively all patients should undergo a systematic mediastinal lymphadenectomy.

Expression of major histocompatibility class I and class II antigens and intercellular adhesion molecule-1 on operable non-small cell lung carcinomas: Frequency and prognostic significance

Major histocompatibility complex (MHC) antigens and adhesion molecules, such as the intercellular adhesion molecule-1 (ICAM-1), appear to play an important role in the immunological recognition and destruction of tumour cells. We, therefore, examined the expression patterns of these proteins on primary tumours of 91 patients with operable non-small cell lung cancer (NSCLC). Applying immunohistochemistry with monoclonal antibody (MAb) W6/32 against a common framework determinant of HLA class I antigens revealed a deficient expression in 33.0% of the cases analysed, while neo-expression of either HLA class II antigens (MAb TAL.1B5) or ICAM-1 (MAb PA3.58-14) was observed in 26.4 or 29.7% of tumours, respectively. Analysis of consecutive tumour specimens indicated that HLA antigens and ICAM-1 were frequently coexpressed. With regard to clinicopathological risk factors, we could demonstrate a preferential expression of those markers in patients with locally restricted and well-differentiated tumours or no lymph node metastases, which was more pronounced in adenocarcinomas than in squamous cell carcinomas. In contrast, the presence versus the absence of HLA antigens and ICAM-1 was not correlated with the rate of tumour recurrence or overall survival in patients with NSCLC. In conclusion, the co-ordinated expression of immunologically relevant cell surface molecules on primary NSCLC is a frequent event that correlates with distinct parameters of favourable prognosis. However, we have no evidence that the immune response facilitated by these molecules can effectively influence the clinical course of the disease.

Expression of MHC molecules and ICAM-1 on non-small cell lung carcinomas: Association with early lymphatic spread of tumour cells

Early microdissemination of tumour cells determines the prognosis of patients with apparently localised non-small cell lung cancer (NSCLC). Monoclonal antibodies to epithelial antigens can now be used to detect single carcinoma cells present in mesenchymal secondary organs such as bone marrow or lymph nodes. The present study was designed to obtain insights into the potential role of the immune system in lymphatic and haematogenous microdissemination of NSCLC cells. Using immunohistochemical staining of primary NSCLC, we assessed the expression pattern of molecules mediating an efficient cellular immune response, that is, MHC class I and class II antigens and the intercellular adhesion molecule-1 (ICAM-1). All 58 patients evaluated were staged as free of overt metastases by conventional clinico-pathological screening. Isolated tumour cells in bone marrow or lymph nodes were identified with mAb CK2 to cytokeratin component No. 18 and mAb BerEp-4 to glycoproteins of 34 and 39 kd present on epithelial cells, respectively. MHC class I expression on primary tumours was reduced or absent in 6/10 (60.0%) patients with isolated cancer cells in lymph nodes as compared to 6/33 tumours (18.1%) without such tumour cell dissemination (P = 0.01). MHC class II molecules on primary tumours were detected in 1/10 (10.0%) patients with micrometastases to regional lymph nodes and in 10/33 (30.3%) patients without such a tumour cell spread. None of the 10 patients with nodal microdissemination expressed ICAM-1 on their primary NSCLC, while such expression was detectable in 12/33 (36.4%) patients without this dissemination (P = 0.01). In contrast, the detection of tumour cells in bone marrow was not correlated to the expression of any of these immunoregulatory molecules. Our data suggest that escape caused by deficient expression of MHC class I antigens and ICAM-1 on tumour cells may support homing or survival of disseminated tumour cells in lymphoid tissue.