Integrated functional and spatial profiling of tumour immune responses induced by immunotherapy: the iPROFILER platform.

BACKGROUND: Cancer immunotherapy elicits functional activation and changes in immune cell distribution in cancer. Tumour heterogeneity is a reason for treatment failure but is difficult to capture in experimental settings. This proof-of-principle study describes the integrated functional and digital spatial profiling platform iPROFILER to capture in-situ immune activation patterns with high precision. MATERIALS AND METHODS: iPROFILER combines an algorithm-based image analysis approach for spatial profiling with functional analyses of patient-derived tumour fragments (PDTFs). This study utilized a folate receptor 1 (FOLR1)xCD3 bispecific antibody in dual-affinity re-targeting (DART) format as a tool for inducing T-cell responses in patient tumour samples, and an in-depth investigation of the immune perturbations induced in the tumour microenvironment was performed. RESULTS: Ex-vivo DART stimulation induces upregulation of multiple activation markers in CD4+ and CD8+ T-cell populations and secretion of pro-inflammatory cytokines in FOLR1-positive tumour specimens. This response was reduced or absent in tissue samples that did not express FOLR1. Immunological responses were driven by a strong induction of interferon gamma (IFNγ) and IFNγ-induced chemokines suggestive of activation of cytotoxic or Th1-like T cells. Ex-vivo DART treatment led to a numerical increase in effector T cells and an upregulation of immune activation markers in the tumour microenvironment as captured by digital image analysis. Analysis of immune activation in tumour and stromal regions further supported the potential of the platform to measure local differences in cell-type-specific activation patterns. CONCLUSIONS: iPROFILER effectively combines functional and spatial readouts to investigate immune responses ex vivo in human tumour samples.

Postoperative deep wound dehiscence of thoracotomy with isolation of Corynebacterium tuberculostearicum: surgical site infection or colonization?

OBJECTIVE: Infections with Corynebacterium tuberculostearicum are very rare as in most of the cases its isolation is associated with tissue colonization rather than infection. CASE REPORT: An 80-year old female patient was sent to the consultation hour of thoracic surgery for evaluation of a symptomatic persistent unilateral pleural effusion of her right lung. The differential diagnosis included either the presence of a chronic pleural empyema or the presence of malignancy. After excluding a malignancy, a decortication of the middle and lower lobe was performed, as the two lobes could not significantly re-expand. The course was further complicated by the presence of two-times deep wound dehiscence, which made necessary a rethoracotomy. The microbiologic results of the biopsies revealed the presence of only Corynebacterium tuberculostearicum with an initially questionable clinical relevance. As soon as the antibiotic treatment for Corynebacterium tuberculostearicum began, together with the use of vacuum-assisted therapy (VAC), the closure of the thoracotomy was accelerated. CONCLUSIONS: Clinically relevant surgical site infections with Corynebacterium species in thoracic surgery are difficult to distinguish. Nevertheless, its combined surgical and antibiotic treatment is warranted when its relevance is questionable due to its resistance to broad-spectrum antibiotics as well as to its potential for the complicated clinical course.

Diffuse lymphatic leakage after continuous vacuum-assisted closure therapy for thoracic wound infection after rib stabilization.

Vacuum-assisted closure (VAC) therapy is a useful tool in the management of a wide spectrum of complex wounds in cardiothoracic surgery. It promotes healing through the application of a controlled and localized negative pressure on porous polyurethane absorbent foams. Known advantages of the VAC therapy are the acceleration of wound healing, stimulation of granulation tissue and reduced tissue edema. Despite its excellent properties, some related complications after and during the therapy have been reported. We report the case of a 47-year-old female with a thoracic wound infection after rib stabilization, managed with open surgery and VAC therapy, which was complicated by a diffuse lymphatic leakage. This is the first case described of diffuse lymphatic leakage followed by partial necrosis of the breast after continuous VAC therapy. We recommend the application of a lower pressure level of this device for complex wounds of the chest wall near the breast.

Prognostic role of FGFR1 amplification in early-stage non-small cell lung cancer.

BACKGROUND: Recently, fibroblast growth factor receptor 1 (FGFR1) was discovered in squamous cell carcinomas (SCC) of the lung with FGFR1 amplification described as a promising predictive marker for anti-FGFR inhibitor treatment. Only few data are available regarding prevalence, prognostic significance and clinico-pathological characteristics of FGFR1-amplified and early-stage non-small cell lung carcinomas (NSCLC). We therefore investigated the FGFR1 gene status in a large number of well-characterised early-stage NSCLC. METHODS: FGFR1 gene status was evaluated using a commercially available fluorescent in situ hybridisation (FISH) probe on a tissue microarray (TMA). This TMA harbours 329 resected, formalin-fixed and paraffin-embedded, nodal-negative NSCLC with a UICC stage I-II. The FISH results were correlated with clinico-pathological features and overall survival (OS). RESULTS: The prevalence of an FGFR1 amplification was 12.5% (41/329) and was significantly (P<0.0001) higher in squamous cell carcinoma (SCC) (20.7%) than in adenocarcinoma (2.2%) and large cell carcinoma (13%). Multivariate analysis revealed significantly (P=0.0367) worse 5-year OS in patients with an FGFR1-amplified NSCLC. CONCLUSIONS: FGFR1 amplification is common in early-stage SCC of the lung and is an independent and adverse prognostic marker. Its potential role as a predictive marker for targeted therapies or adjuvant treatment needs further investigation.

Prospective study of a panfungal PCR assay followed by sequencing, for the detection of fungal DNA in normally sterile specimens in a clinical setting: a complementary tool in the diagnosis of invasive fungal disease?

We prospectively analyzed 34 clinical biopsy samples from 23 patients with a suspected invasive fungal infection by fungal culture, histology and a panfungal PCR followed by sequencing. Results were compared to the composite diagnosis according the European Organization for Research and Treatment of Cancer (EORTC) criteria. In 34 samples, culture, histology and panfungal PCR were positive in 35%, 38% and 62%, respectively. On the sample level the panfungal PCR revealed a sensitivity of 69% and a specificity of 62.5% compared to proven IFI according postoperative EORTC criteria. On patient level, the sensitivity of the PCR approach was 100%, specificity 62.5%.

[Surgical operability of patients with locally advanced non-small cell lung cancer (NSCLC)]. / Lungenresektionen beim nicht-kleinzelligen Bronchuskarzinom - wann ist auch ein maximalextensiver Ansatz gerechtfertigt?

Local advanced NSCLC are not rare and consist of a heterogeneous group of diseases. Surgery can be offered to high selected patients. In most of the cases, the therapy concept is multidisciplinary, combining surgery with chemotherapy, radiotherapy, or both of them. Such a concept, in which surgery plays a central role, is very often the only way to treat the patient in a curative intention. Unfortunately, in T4-tumors, distant metastases and an extensive involvement of the mediastinal lymph nodes are already present at the time of diagnosis in most of the cases. In such palliative situations, surgery is not indicated. The most important factor to select patients for surgery is the possibility to perform a radical complete resection of the tumor. The standard resection consists of an en-bloc resection of lung tissue with the infiltrated neighbouring structure. The prognosis depends on the radicality of the resection and of the lymph node status. The best results are observed in patients with isolated infiltration of the chest wall, with tumors of the sulcus superior, and infiltration of the superior vena cava. 5-y overall survival up to 40 - 50% can be observed after complete resection in N0-patients. The prognosis is poorer in patients with infiltration of the carina, of the spine and of the atrium, with 20 - 30% in small series. Local advanced NSCLC should only be treated in reference centres. Only in such centres, the interdisciplinary approach and the optimal therapy can be guaranteed with a minimal morbidity and mortality.

Cigarette smoke inhibits lung fibroblast proliferation by translational mechanisms.

Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD) and emphysema. Although cigarette smoke represses cellular proliferation, the molecular mechanisms underlying this phenomenon are unknown. CCAAT/enhancer-binding proteins (C/EBPs) are key regulators of cell cycle progression, differentiation and pro-inflammatory gene expression, are regulated predominantly at the translational level and may be involved in the pathogenesis of COPD. The aim of this study was to assess the effect of cigarette smoke on proliferation and the expression and translational regulation of C/EBPα and C/EBPß in nondiseased primary human lung fibroblasts. Fibroblasts were exposed to cigarette smoke-conditioned medium (10% and 20% for 24 h). Proliferation was determined by [(3)H]thymidine incorporation. Protein expression levels were determined by immunoblotting and translation was monitored using a translation control reporter system. Cigarette smoke significantly reduced fibroblast proliferation and significantly upregulated full-length C/EBPα and C/EBPß proteins due to a shift in the translational control of CEBPA and CEBPB mRNAs. This shift involved the re-initiation of mRNA translation via the regulatory upstream open reading frame, which coincided with increased interleukin-8 release and a decrease in functional elastin level. These findings provide a novel mechanism to understanding the tissue remodelling observed in the lungs of COPD patients.

Accuracy of CT parameters for assessment of tumour size and aggressiveness in lung adenocarcinoma with bronchoalveolar elements.

Accurate determination of tumour size in lung adenocarcinoma with bronchoalveolar features (BAC) is important for the determination of TNM (tumour, nodes, metastasis) scores used in staging, prognosis and therapy response assessment. However, tumour sizes derived using lung window (LW) CT or soft-tissue/mediastinal window (MW) CT often give different results. This study examines which measurement correlates best with actual tumour size and which best identifies advanced disease. This retrospective study included 43 BAC patients who underwent surgical resection with mediastinal lymphadenectomy <4 weeks post CT scan. The largest unidimensional tumour diameter on each CT window was compared with actual histopathological tumour size (HP). LW, MW and HP size measurements and a recently described CT parameter - the modified tumour shadow disappearance rate (mTDR) = (1 - [MW/LW]) - were then used to determine which parameter best discriminated between the presence or absence of advanced disease. There was no difference between HP and LW sizes, but MW significantly underestimated HP size (p<0.0001). Unlike MW (p = 0.01) and mTDR (p = 0.001), neither HP (p = 0.14) nor LW (p = 0.10) distinguished between patients with or without advanced disease. On receiver operating characteristic (ROC) analysis at a cut-off of ≤0.13, the sensitivity and specificity of mTDR for detecting advanced disease were 69% and 89%, respectively. In patients with tumours ≤3 cm, only mTDR remained a significant predictor of advanced disease (p = 0.017), with best cut-off at ≤0.20, giving a sensitivity and specificity of 71% and 94%, respectively. MW better predicts advanced disease than LW and might also need to be recorded for RECIST (response evaluation criteria in solid tumours) assessment for T staging of BAC; however, mTDR appears to be an even better predictor and should also be used.

Autologous fibrin sealant reduces the incidence of prolonged air leak and duration of chest tube drainage after lung volume reduction surgery: a prospective randomized blinded study.

OBJECTIVE: Prolonged air leak is reported in up to 50% of patients after lung volume reduction surgery. The effect of an autologous fibrin sealant on the intensity and duration of air leak and on the time to chest drain removal after lung volume reduction surgery was investigated in a randomized prospective clinical trial. METHODS: Twenty-five patients underwent bilateral thoracoscopic lung volume reduction surgery. In each patient, an autologous fibrin sealant was applied along the staple lines on one side, whereas no additional measure was taken on the other side. Randomization of treatment was performed at the end of the resection on the first side. Air leak was assessed semiquantitatively by use of a severity score (0 = no leak; 4 = continuous severe leak) by two investigators blinded to the treatment. RESULT: Mean value of the total severity scores for the first 48 hours postoperative was significantly lower in the treated group (4.7 +/- 7.7) than in the control group (16.0 +/- 10.1) (P < .001), independently of the length of the resection. Prolonged air leak and mean duration of drainage were also significantly reduced after application of the sealant (4.5% and 2.8 +/- 1.9 days versus 31.8% and 5.9 +/- 2.9 days) (P = .03 and P < .001). CONCLUSIONS: Autologous fibrin sealant for reinforcement of the staple lines after lung volume reduction surgery significantly reduces prolonged air leak and duration of chest tube drainage.

Multicenter trial of neo-adjuvant chemotherapy followed by extrapleural pneumonectomy in malignant pleural mesothelioma.

BACKGROUND: The aim of this multicenter trial was to prospectively evaluate neo-adjuvant chemotherapy followed by extrapleural pneumonectomy (EPP) and radiotherapy, including quality of life as outcome. PATIENTS AND METHODS: Eligible patients had malignant pleural mesothelioma of all histological types, World Health Organization performance status of zero to two and clinical stage T1-T3, N0-2, M0 disease considered completely resectable. Neo-adjuvant chemotherapy consisted of three cycles of cisplatin and gemcitabine followed by EPP. Postoperative radiotherapy was considered for all patients. RESULTS: In all, 58 of 61 patients completed three cycles of neo-adjuvant chemotherapy. Forty-five patients (74%) underwent EPP and in 37 patients (61%) the resection was complete. Postoperative radiotherapy was initiated in 36 patients. The median survival of all patients was 19.8 months [95% confidence interval (CI) 14.6-24.5]. For the 45 patients undergoing EPP, the median survival was 23 months (95% CI 16.6-32.9). Psychological distress showed minor variations over time with distress above the cut-off score indicating no morbidity with 82% (N = 36) at baseline and 76% (N = 26) at 3 months after surgery (P = 0.5). CONCLUSIONS: The observed rate of operability is promising. A median survival of 23 months for patients undergoing EPP compares favourably with the survival reported from single center studies of upfront surgery. This approach was not associated with an increase in psychological distress.

European trends in preoperative and intraoperative nodal staging: ESTS guidelines.

Preoperative and intraoperative lymph node (LN) staging is of paramount importance for patients with non-small cell lung cancer. The Council of the European Society of Thoracic Surgery took the initiative to organize workshops on intraoperative and preoperative mediastinal LN staging. This resulted in specific guidelines. Relevant peer-reviewed publications on these subjects, the experience of the participants, and the opinion of the European Society of Thoracic Surgery members contributing online were used to reach a consensus. For primary staging, mediastinoscopy remains the gold standard for the superior mediastinal LNs. Invasive procedures can be omitted in patients with peripheral tumors and negative mediastinal and hilar nodes on positron emission tomography scan. Positron emission tomography-positive mediastinal findings should always be cytohistologically confirmed. New minimally invasive techniques that provide cytohistological diagnosis became available. Their specificity is high, but the negative predictive value is low. If they yield negative results, an invasive surgical technique remains indicated. For restaging, invasive techniques providing cytohistological information are advisable. Systematic nodal dissection is recommended in all cases to ensure complete resection. Lobe-specific systematic nodal dissection is acceptable for peripheral squamous T1 tumors if hilar and interlobar nodes are negative on frozen section studies. The report from the pathologist should describe the number of LNs removed and studied, the overall number of metastatic LNs in each station, and the status of the LN capsule. We hope that the adherence to these guidelines will standardize and improve preoperative and intraoperative LN staging and pathologic evaluation of non-small cell lung cancer.

Taurolidine and povidone-iodine induce different types of cell death in malignant pleural mesothelioma.

Taurolidine and povidone-iodine (PVP-I) are used in every day clinical practice, taurolidine as a broad spectrum antibiotic, and PVP-I as an antiseptic. The type of cell death induced in malignant pleural mesothelioma (MPM) cell lines by these agents was compared, and their ability to sensitize to chemotherapy assessed. Both taurolidine and PVP-I inhibited MPM cell growth after 7.5min incubation, but taurolidine was more effective at later time points and was more specific towards tumour cells than PVP-I. Taurolidine induced death by caspase-dependent and independent mechanisms, whereas in contrast, PVP-I induced a necrotic phenotype that was not caspase-dependent. Interestingly, both taurolidine and PVP-I induced the production of reactive oxygen intermediates and decreased mitochondrial membrane permeability, and cell death was inhibited by the oxygen scavenger N-acetyl cysteine. Taurolidine but not PVP-I treatment resulted in p53 activation in 2/3 MPM cell lines and a decrease in the protein levels of survivin, Bcl-2 and Mcl-1. Survivin also decreased in response to PVP-I whereas Bcl-xL remained unaffected by both treatments. Targeting of Bcl-xL with siRNA sensitized MPM cells to taurolidine and taurolidine treatment sensitized MPM cells to cisplatin-induced apoptosis. In conclusion, taurolidine and PVP-I are both cytotoxic to human MPM cells at early and late time points and induce reactive oxygen intermediate production. Taurolidine induces apoptosis and necrosis, activates p53 and sensitizes cells to cisplatin, whereas PVP-I inhibits cell growth via necrosis. Both agents are promising candidates for use in local treatment within multimodality concepts for MPM.

Intrapleural topical application of cisplatin with the surgical carrier Vivostat increases the local drug concentration in an immune-competent rat model with malignant pleuromesothelioma.

OBJECTIVE: We sought to investigate whether intrapleural topical application of cisplatin with a surgical carrier has a prolonged local tissue level in comparison with cisplatin solution while reducing systemic toxicity. METHODS: Forty immune-competent Fischer rats were inoculated with 10(6) mesothelioma cells. Ten days later, left pneumonectomy with tumor debulking was performed. Twenty animals underwent local application of cisplatin solution (100 mg/m2), whereas the same quantity of cisplatin was topically applied as a gel with the Vivostat (Vivolution) system in 20 other animals. In each group 5 subgroups of 4 animals were defined according to the harvesting time of blood and tissue samples (2, 4, 24, and 72 hours and 1 week) after local therapy. Platinum concentrations in serum and tissue and systemic toxicity were analyzed. RESULTS: Platinum concentrations in tissue were significantly higher in the gel group (group 1) than in the solution group (group 2) at 1, 3, and 7 days after therapy (1510, 1224, and 1069 pg/mg for group 1 vs 598, 382, and 287 pg/mg for group 2; P = .007, P = .005, and P = .0002, respectively). Laboratory findings showed renal insufficiency in the animals of the solution group at 1 week, with values of 98 mmol/L versus 7.7 mmol/L for urea and 410 mumol/L versus 43 mumol/L for creatinine (P = .02 and P = .01, respectively), which was confirmed by means of pathologic analysis. CONCLUSIONS: Intrapleural administration of cisplatin with the carrier Vivostat significantly provides sustained higher platinum concentrations up to 1 week in tissue in comparison with application of cisplatin solution without conferring systemic toxicity in this model.

Surgery for non-localized bronchiectasis.

BACKGROUND: Resection for localized bronchiectasis is a well established therapy. However, there is little information on the role of surgery in non-localized bronchiectasis. METHODS: Between January 1992 and April 2001, 55 patients without cystic fibrosis underwent resection. Forty-eight patients (mean age 45 (range 23-74) years; 32 women) were available for long-term follow-up. Twenty-five patients underwent resection for localized disease (group 1) and 23 had bronchiectasis in at least two different lobes (group 2). RESULTS: Thirty-one of the 48 patients were treated by Video Assisted Thoracoscopic Surgery (VATS) resection. There was no 30-day mortality. Mean duration of hospital stay was 10.9 (range 6-31) days in group 1 and 11.1 (range 5-19) days in group 2. Three of 25 patients in group 1 required reoperation. Only minor complications occurred in group 2 (three patients). Mean follow-up for both groups was 37 (range 6-97) months. Twenty-three of 25 patients in group 1 and 16 of 23 in group 2 reported satisfaction at 6 months after the operation. Recurrent infection was noted in three patients in each group. Haemoptysis recurred in only one patient in group 2. CONCLUSION: The surgical treatment of selected patients with non-localized bronchiectasis was safe and most patients were satisfied with the outcome.

Non-steroidal anti-inflammatory drugs decrease the quality of pleurodesis after mechanical pleural abrasion.

OBJECTIVE: Non-steroidal anti-inflammatory drugs (NSAIDs) are often applied for pain management after thoracic surgery. Since these drugs diminish collagen deposition through inhibition of the prostaglandin synthesis, we investigated their effects on adhesion formation after endoscopic mechanical pleural abrasion, which is often applied in the therapy of pneumothorax. METHODS: Mechanical pleural abrasion was performed unilaterally by the use of video-assisted thoracoscopic surgery technique in an established pig model. Ten animals (41.3+/-3.4 kg) were divided into a treatment group and a control group. In the treatment group, animals received 100 mg diclofenac (2 mg/kg body weight) orally daily for 3 weeks after surgery. At 3 weeks, all animals were sacrificed and efficacy of pleurodesis was macroscopically assessed by three independent reviewers blinded to the treatment of animals using a five-point severity pleurodesis score (from 0, no adhesions to 4, complete symphisis) and obliteration grade rating the distribution of adhesions (from 0, no adhesions to 4, adhesions in the whole chest). Microscopic evaluation was performed by two pathologists blinded to the study groups as well. A four-point score assessed the amount of collagen deposition (from 1, a few collagen fibers to 4, scar). RESULTS: Gross observation showed more dense adhesions in control animals with a median pleurodesis score of 3.67+/-1.0 in comparison to 2+/-2.2 in the treatment group (P = 0.01 *, Mann-Whitney non-parametric test). Distribution of adhesions was comparable in both groups with a median obliteration score of 3.67+/-1.3. Histopathologic examination showed a higher amount of collagen deposition in the control group, suggesting more dense adhesions, whereas in the treatment group there was loose granulation tissue (score of 4.0+/-0.8 vs. 2.3+/-1.0 in the treatment group, P = 0.06). The degree of inflammatory reaction was comparable in the two groups. CONCLUSIONS: Our results demonstrate that perioperative use of NSAIDs highly affects the quality of pleural adhesions obtained after mechanical abrasion in this pig model, which further suggests that these drugs should be avoided for pain management when a pleurodesis is performed.

Bronchoscopic radioisotope injection for sentinel lymph-node mapping in potentially resectable non-small-cell lung cancer.

OBJECTIVE: Prospective study to evaluate the feasibility of a preoperative bronchoscopic radioisotope application, followed by conventional sentinel lymph-node (SLN) identification and to investigate the occurrence and distribution of micrometastases in relation to SLN activity. METHODS: Twenty patients with a mean age of 63 years and proven clinical stage T1-3 N0-1 non-small-cell lung cancer (NSCLC) were included. A dosage of 80MBq radiolabeled technetium-99m nanocolloid was endoscopically administrated on intubated patients in the operation theatre. At thoracotomy, scintigraphic readings of both the primary tumor and hilar and mediastinal lymph-node stations were obtained with a hand-held gamma-counter. Patients underwent lung resection and mediastinal lymphadenectomy. Radiolabeled nodes were also examined separately on back-table. SLNs were defined as the hottest nodes or nodes with at least one-tenth of the radioactivity of the hottest nodes. SLNs pathologic assessment included standard examination using hematoxylin and eosin staining on step sections and immunohistochemistry (ICH) for cytokeratins. RESULTS: Identification of SLNs was possible in 19/20 (95%) patients after bronchoscopic radioisotope application. In 7/19 (37%) patients, a unique SLN was identified, whereas in 12/19 (63%) patients, nodes from two different stations could be classified as SLNs. Metastatic nodal disease was found in 9/19 (47%) patients. ICH revealed micrometastases in 2/12 (17%) patients, initially classified nodal negative. Pathologic negative SLNs were a predictor for absence of metastatic nodal disease after mediastinal lymphadenectomy. No complication related to the procedure was observed. CONCLUSION: Our preliminary results suggest that preoperative bronchoscopic radioisotope injection for SLN identification is a safe and simple method, improving accuracy of SLN detection in comparison to intraoperative technique. The absence of metastases in the SLNs seems to predict a negative nodal status accurately.

Morbidity and outcome after sentinel lymph node dissection in patients with early-stage malignant cutaneous melanoma.

OBJECTIVE: Prospective analysis of the morbidity and outcome of the sentinel lymph node (SLN) technique in a consecutive series of patients with early-stage melanoma. METHODS: Between 1997 and 1998, 60 patients with stage IB-II malignant melanoma underwent SLN dissection. Preoperative dynamic lymphoscintigraphy with mapping of the lymph vessels and lymph nodes and location of the sentinel node was performed the day before SLN dissection. SLN was identified by use of the blue dye technique. SLN was assessed for histopathological and immunohistochemical examination. Postoperative morbidity and mortality were recorded. Follow-up consisted of repetitive clinical examination with lymph nodes status, laboratory and radiologic findings. RESULTS: Tumor-positive SLN was observed in 18% of the patients and stage II disease was found in 91% of the patients with positive SLN. Breslow thickness was the only significant factor predicting involvement of a SLN (p = 0.02). In 36% of the positive SLN, metastases could be assessed only by immunohistochemical examination. Postoperative complications after SLN dissection were observed in 5% in comparison with 36% after elective lymph node dissection. After a mean follow-up of 32 months, recurrence was observed in 3% with a mean disease-free survival of 8 months. Overall survival was 82% and 90% in patients with positive and negative SLN, respectively. Overall mortality was 15%, due to distant metastases in 78% of the cases. CONCLUSIONS: Staging of early-stage melanoma with the SLN dissection by use of the blue dye technique combined to lymphoscintigraphy and immunohistochemistry is reliable and safe, with less morbidity than elective lymphadenectomy. Long-term follow-up is mandatory to establish the exact reliability of SLN dissection.

Alterations of cell cycle regulators are less frequent in advanced non-small cell lung cancer than in resectable tumours.

Prognosis of lung cancer is related to stage of disease at time of diagnosis. In this study we examine alterations of pathways governing the cell cycle, in particular pRb-cyclinD1-p16 alpha and p53-p14ARF, in a series of NSCLC (n=92) at different stages at diagnosis. Using immunohistochemistry, we assessed the expression of the retinoblastoma protein (pRb), cyclin D1, p16 alpha, p53 and p14ARF. Tumours in stage I-IIIA (resectable) were more likely to have alterations in the pRb-cyclinD1-p16 alpha pathway than tumours in advanced stage (IIIB-IV) (90% versus 63%, P=0.002). pRb and p14ARF were more frequently downregulated in resectable tumours (P< or =0.03), and cyclin D1, p16 alpha, and p53 were altered at a similar frequency in resectable and advanced tumours. In 12 patients, metastatic sites (5 lymph node, 3 bone, 2 brain and 2 gastrointestinal metastases) were available for comparison with the primary tumour: 19 altered protein expressions were found to be concordant, six additional alterations (in 4 patients) were found in the metastases only, especially in lymph node metastases (3 patients). Compared with normal protein expression, both pathway alterations were associated with a longer survival (P=0.02). In a multivariate analysis (Cox regression) this difference was not maintained after adjustment for age, stage and tumour differentiation. Cyclin D1 was the sole protein with independent prognostic value in resectable tumours: the relative risk of local relapse was 4.7 in tumours without cyclin D1 overexpression (P=0.02, Cox regression analysis). No protein studied had a predictive significance for response after chemotherapy in non-resectable tumours. These results demonstrate a strong correlation between stage and pathway alterations, cell cycle regulators being less likely altered in advanced NSCLC. Tumours with defects in these control pathways tend therefore to remain localised and to metastasize at a later phase in tumour development. This finding might be an explanation for distinct biological behaviour (e.g. chemotherapy response) of resectable versus advanced disease.