Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

Single-agent versus combination chemotherapy in patients with advanced non-small cell lung cancer and a performance status of 2 : prognostic factors and treatment selection based on two large randomized clinical trials

Purpose: Data from two randomized phase III trials were analyzed to evaluate prognostic factors and treatment selection in the first-line management of advanced non-small cell lung cancer patients with performance status (PS) 2. Patients and Methods: Patients randomized to combination chemotherapy (carboplatin and paclitaxel) in one trial and single-agent therapy (gemcitabine or vinorelbine) in the second were included in these analyses. Both studies had identical eligibility criteria and were conducted simultaneously. Comparison of efficacy and safety was performed between the two cohorts. A regression analysis identified prognostic factors and subgroups of patients that may benefit from combination or single-agent therapy. Results: Two hundred one patients were treated with combination and 190 with single-agent therapy. Objective responses were 37 and 15%, respectively. Median time to progression was 4.6 months in the combination arm and 3.5 months in the single-agent arm (p < 0.001). Median survival imes were 8.0 and 6.6 months, and 1-year survival rates were 31 and 26%, respectively. Albumin <3.5 g, extrathoracic metastases, lactate dehydrogenase ≥200 IU, and 2 comorbid conditions predicted outcome. Patients with 0-2 risk factors had similar outcomes independent of treatment, whereas patients with 3-4 factors had a nonsignificant improvement in median survival with combination chemotherapy. Conclusion: Our results show that PS2 non-small cell lung cancer patients are a heterogeneous group who have significantly different outcomes. Patients treated with first-line combination chemotherapy had a higher response and longer time to progression, whereas overall survival did not appear significantly different. A prognostic model may be helpful in selecting PS 2 patients for either treatment strategy. © 2009 by the International Association for the Study of Lung Cancer.

Epigenetic regulation of glucose transporters in non-small cell lung cancer

Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Combination therapy with gefitinib and rofecoxib in patients with platinum-pretreated relapsed non-small-cell lung cancer

Purpose: In non-small-cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) play major roles in tumorigenesis. This phase I/II study evaluated combined therapy with the EGFR tyrosine kinase inhibitor (TKI) gefitinib and the COX-2 inhibitor rofecoxib in platinum-pretreated, relapsed, metastatic NSCLC (n = 45). Patients and Methods: Gefitinib 250 mg/d was combined with rofecoxib (dose escalated from 12.5 to 25 to 50 mg/d through three cohorts, each n = 6). Because the rofecoxib maximum-tolerated dose was not reached, the 50 mg/d cohort was expanded for efficacy evaluation (n = 33). Results: Among the 42 assessable patients, there was one complete response (CR) and two partial responses (PRs) and 12 patients with stable disease (SD); disease control rate was 35.7% (95% CI, 21.6% to 52.0%). Median time to tumor progression was 55 days (95% CI, 47 to 70 days), and median survival was 144 days (95% CI, 103 to 190 days). In a pilot study, matrix-assisted laser desorption/ionization (MALDI) proteomics analysis of baseline serum samples could distinguish patients with an objective response from those with SD or progressive disease (PD), and those with disease control (CR, PR, and SD) from those with PD. The regimen was generally well tolerated, with predictable toxicities including skin rash and diarrhea. Conclusion: Gefitinib combined with rofecoxib provided disease control equivalent to that expected with single-agent gefitinib and was generally well tolerated. Baseline serum proteomics may help identify those patients most likely to benefit from EGFR TKIs. © 2007 by American Society of Clinical Oncology.

Molecular biomarkers in non-small-cell lung cancer : a retrospective analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 FLEX study showed that the addition of cetuximab to cisplatin and vinorelbine significantly improved overall survival, compared with cisplatin and vinorelbine alone, in the first-line treatment of EGFR-expressing, advanced non-small-cell lung cancer (NSCLC). We investigated whether candidate biomarkers were predictive for the efficacy of chemotherapy plus cetuximab in this setting. Methods: Genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour tissue of patients enrolled in the FLEX study was screened for KRAS codon 12 and 13 and EGFR kinase domain mutations with PCR-based assays. In FFPE tissue sections, EGFR copy number was assessed by dual-colour fluorescence in-situ hybridisation and PTEN expression by immunohistochemistry. Treatment outcome was investigated according to biomarker status in all available samples from patients in the intention-to-treat population. The primary endpoint in the FLEX study was overall survival. The FLEX study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT00148798. Findings: KRAS mutations were detected in 75 of 395 (19%) tumours and activating EGFR mutations in 64 of 436 (15%). EGFR copy number was scored as increased in 102 of 279 (37%) tumours and PTEN expression as negative in 107 of 303 (35%). Comparisons of treatment outcome between the two groups (chemotherapy plus cetuximab vs chemotherapy alone) according to biomarker status provided no indication that these biomarkers were of predictive value. Activating EGFR mutations were identified as indicators of good prognosis, with patients in both treatment groups whose tumours carried such mutations having improved survival compared with those whose tumours did not (chemotherapy plus cetuximab: median 17·5 months [95% CI 11·7-23·4] vs 8·5 months [7·1-10·8], hazard ratio [HR] 0·52 [0·32-0·84], p=0·0063; chemotherapy alone: 23·8 months [15·2-not reached] vs 10·0 months [8·7-11·0], HR 0·35 [0·21-0·59], p<0·0001). Expression of PTEN seemed to be a potential indicator of good prognosis, with patients whose tumours expressed PTEN having improved survival compared with those whose tumours did not, although this finding was not significant (chemotherapy plus cetuximab: median 11·4 months [8·6-13·6] vs 6·8 months [5·9-12·7], HR 0·80 [0·55-1·16], p=0·24; chemotherapy alone: 11·0 months [9·2-12·6] vs 9·3 months [7·6-11·9], HR 0·77 [0·54-1·10], p=0·16). Interpretation: The efficacy of chemotherapy plus cetuximab in the first-line treatment of advanced NSCLC seems to be independent of each of the biomarkers assessed. Funding: Merck KGaA. © 2011 Elsevier Ltd.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.

Prognostic factors in patients with advanced non-small cell lung cancer : data from the phase III FLEX study

The FLEX study demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). In the FLEX intention to treat (ITT) population, we investigated the prognostic significance of particular baseline characteristics. Individual patient data from the treatment arms of the ITT population of the FLEX study were combined. Univariable and multivariable Cox regression models were used to investigate variables with potential prognostic value. The ITT population comprised 1125 patients. In the univariable analysis, longer median survival times were apparent for females compared with males (12.7 vs 9.3 months); patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 compared with 1 compared with 2 (13.5 vs 10.6 vs 5.9 months); never smokers compared with former smokers compared with current smokers (14.6 vs 11.1 vs 9.0); Asians compared with Caucasians (19.5 vs 9.6 months); patients with adenocarcinoma compared with squamous cell carcinoma (12.4 vs 9.3 months) and those with metastases to one site compared with two sites compared with three or more sites (12.4 months vs 9.8 months vs 6.4 months). Age (<65 vs ≥65 years), tumor stage (IIIB with pleural effusion vs IV) and percentage of tumor cells expressing EGFR (<40% vs ≥40%) were not identified as possible prognostic factors in relation to survival time. In multivariable analysis, a stepwise selection procedure identified age (<65 vs ≥65 years), gender, ECOG PS, smoking status, region, tumor histology, and number of organs involved as independent factors of prognostic value. In summary, in patients with advanced NSCLC enrolled in the FLEX study, and consistent with previous analyses, particular patient and disease characteristics at baseline were shown to be independent factors of prognostic value. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. © 2012 Elsevier Ireland Ltd.

Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX) : an open-label randomised phase III trial

Background: Use of cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), has the potential to increase survival in patients with advanced non-small-cell lung cancer. We therefore compared chemotherapy plus cetuximab with chemotherapy alone in patients with advanced EGFR-positive non-small-cell lung cancer. Methods: In a multinational, multicentre, open-label, phase III trial, chemotherapy-naive patients (≥18 years) with advanced EGFR-expressing histologically or cytologically proven stage wet IIIB or stage IV non-small-cell lung cancer were randomly assigned in a 1:1 ratio to chemotherapy plus cetuximab or just chemotherapy. Chemotherapy was cisplatin 80 mg/m 2 intravenous infusion on day 1, and vinorelbine 25 mg/m 2 intravenous infusion on days 1 and 8 of every 3-week cycle) for up to six cycles. Cetuximab-at a starting dose of 400 mg/m 2 intravenous infusion over 2 h on day 1, and from day 8 onwards at 250 mg/m 2 over 1 h per week-was continued after the end of chemotherapy until disease progression or unacceptable toxicity had occurred. The primary endpoint was overall survival. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Between October, 2004, and January, 2006, 1125 patients were randomly assigned to chemotherapy plus cetuximab (n=557) or chemotherapy alone (n=568). Patients given chemotherapy plus cetuximab survived longer than those in the chemotherapy-alone group (median 11·3 months vs 10·1 months; hazard ratio for death 0·871 [95% CI 0·762-0·996]; p=0·044). The main cetuximab-related adverse event was acne-like rash (57 [10%] of 548, grade 3). Interpretation: Addition of cetuximab to platinum-based chemotherapy represents a new treatment option for patients with advanced non-small-cell lung cancer. Funding: Merck KGaA. © 2009 Elsevier Ltd. All rights reserved.

EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer : analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 First-Line ErbituX in lung cancer (FLEX) study showed that the addition of cetuximab to first-line chemotherapy significantly improved overall survival compared with chemotherapy alone (hazard ratio [HR] 0·871, 95% CI 0·762-0·996; p=0·044) in patients with advanced non-small-cell lung cancer (NSCLC). To define patients benefiting most from cetuximab, we studied the association of tumour EGFR expression level with clinical outcome in FLEX study patients. Methods: We used prospectively collected tumour EGFR expression data to generate an immunohistochemistry score for FLEX study patients on a continuous scale of 0-300. We used response data to select an outcome-based discriminatory threshold immunohistochemistry score for EGFR expression of 200. Treatment outcome was analysed in patients with low (immunohistochemistry score <200) and high (≥200) tumour EGFR expression. The primary endpoint in the FLEX study was overall survival. We analysed patients from the FLEX intention-to-treat (ITT) population. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Tumour EGFR immunohistochemistry data were available for 1121 of 1125 (99·6%) patients from the FLEX study ITT population. High EGFR expression was scored for 345 (31%) evaluable patients and low for 776 (69%) patients. For patients in the high EGFR expression group, overall survival was longer in the chemotherapy plus cetuximab group than in the chemotherapy alone group (median 12·0 months [95% CI 10·2-15·2] vs 9·6 months [7·6-10·6]; HR 0·73, 0·58-0·93; p=0·011), with no meaningful increase in side-effects. We recorded no corresponding survival benefit for patients in the low EGFR expression group (median 9·8 months [8·9-12·2] vs 10·3 months [9·2-11·5]; HR 0·99, 0·84-1·16; p=0·88). A treatment interaction test assessing the difference in the HRs for overall survival between the EGFR expression groups suggested a predictive value for EGFR expression (p=0·044). Interpretation: High EGFR expression is a tumour biomarker that can predict survival benefit from the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC. Assessment of EGFR expression could offer a personalised treatment approach in this setting. Funding: Merck KGaA. © 2012 Elsevier Ltd.

Randomized phase II study of cetuximab plus cisplatin/vinorelbine compared with cisplatin/vinorelbine alone as first-line therapy in EGFR-expressing advanced non-small-cell lung cancer

Background: The Lung Cancer Cetuximab Study is an open-label, randomized phase II pilot study of cisplatin and vinorelbine combined with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody cetuximab versus cisplatin and vinorelbine alone, in patients with advanced EGFR-expressing, non-small-cell lung cancer (NSCLC). End points of the study are activity, safety and pharmacokinetics. Patients and methods: Following randomization, for a maximum of eight cycles, patients received three-weekly cycles of cisplatin (80 mg/m2, day 1) and vinorelbine (25 mg/m2 on days 1 and 8) alone or following cetuximab treatment (initial dose 400 mg/m, followed by 250 mg/m2 weekly thereafter). Results: Eighty-six patients were randomly allocated to the study (43 per arm). Confirmed response rates were 28% in the cisplatin/vinorelbine arm (A) and 35% in the cetuximab plus cisplatin/vinorelbine arm (B). Median progression-free survival (PFS) was 4.6 months in arm A and 5.0 months in arm B, with PFS rates at 12 months of 0% and 15%, respectively. Median survival was 7.3 months in arm A and 8.3 months in arm B. The 24-month survival rates were 0% and 16%, respectively. The cetuximab combination was well tolerated. Conclusion: In the first-line treatment of advanced NSCLC, the combination of cetuximab plus cisplatin/vinorelbine demonstrated an acceptable safety profile and the potential to improve activity over cisplatin/vinorelbine alone. © 2007 European Society for Medical Oncology.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

Phospho-Akt expression is associated with a favorable outcome in non-small cell lung cancer

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037). These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC. © 2005 American Association for Cancer Research.

Treatment rationale study design for the MetLung trial : a randomized, double-blind phase III study of onartuzumab (MetMAb) in combination with erlotinib versus erlotinib alone in patients who have received standard chemotherapy for stage IIIB or IV met-positive non-small-cell lung cancer

We present the treatment rationale and study design of the MetLung phase III study. This study will investigate onartuzumab (MetMAb) in combination with erlotinib compared with erlotinib alone, as second- or third-line treatment, in patients with advanced non-small-cell lung cancer (NSCLC) who are Met-positive by immunohistochemistry. Approximately 490 patients (245 per treatment arm) will receive erlotinib (150 mg oral daily) plus onartuzumab or placebo (15 mg/kg intravenous every 3 weeks) until disease progression, unacceptable toxicity, patient or physician decision to discontinue, or death. The efficacy objectives of this study are to compare overall survival (OS) (primary endpoint), progression-free survival, and response rates between the 2 treatment arms. In addition, safety, quality of life, pharmacokinetics, and translational research will be investigated across treatment arms. If the primary objective (OS) is achieved, this study will provide robust results toward an alternative treatment option for patients with Met-positive second- or third-line NSCLC. © 2012 Elsevier Inc. All Rights Reserved.

Tumour necrosis is an independent prognostic marker in non-small cell lung cancer : correlation with biological variables

Background Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. Methods In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. Results Reproducibility was attained in 36/40 cases, Kappa score=0.8 P<0.001. TN correlated with T-stage (P=0.001), platelet count (P=0.004) and p53 expression (P=0.031). Near significant associations of TN with N-stage (P=0.063) and MMP-9 expression (P=0.058) were seen. No association was found with angiogenesis (P=0.98). On univariate (P=0.0016) and multivariate analysis (P=0.023) TN was prognostic. Conclusion These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved.