Patterns of somatic mutation in human cancer genomes.

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.

Non-small-cell lung cancer.

In the decade since the last Lancet Seminar on lung cancer there have been advances in many aspects of the classification, diagnosis, and treatment of non-small-cell lung cancer (NSCLC). An international panel of experts has been brought together to focus on changes in the epidemiology and pathological classification of NSCLC, the role of CT screening and other techniques that could allow earlier diagnosis and more effective treatment of the disease, and the recently introduced seventh edition of the TNM classification and its relation to other prognostic factors such as biological markers. We also describe advances in treatment that have seen the introduction of a new generation of chemotherapy agents, a proven advantage to adjuvant chemotherapy after complete resection for specific stage groups, new techniques for the planning and administration of radiotherapy, and new surgical approaches to assess and reduce the risks of surgical treatment.

The new tumor, node, and metastasis staging system.

The seventh edition of the tumor, node, and metastasis (TNM) classification is based on the proposals of the Staging Project of the International Association for the Study of Lung Cancer (IASLC). The analyses of the IASLC international database of 81,015 patients diagnosed with lung cancer between 1990 and 2000 were used to validate the TNM descriptors. The changes include: the subclassification of T1 and T2 tumors into T1a (≤ 2 cm) and T1b (>2 and ≤ 3 cm), and T2a (>3 and ≤ 5 cm) and T2b (>5 and ≤ 7 cm), respectively; the reclassification of T2 tumors >7 cm as T3; the reclassification of T4 tumors by additional nodules in the same lobe of the primary tumor as T3; the reclassification of M1 tumors by additional nodules in another ipsilateral lobe as T4; the reclassification of pleural and pericardial dissemination, and contralateral M1 nodules as M1a; and the separation of intrathoracic (M1a) and extrathoracic (M1b) metastases. Other innovations include the emphasis on the use of the TNM classification for small cell carcinoma, the inclusion of bronchopulmonary carcinoids into this staging system, the proposal of a new lymph node map, and the adoption of a new, internationally agreed definition of visceral pleura invasion. All these changes improve the separation of tumors with significantly different prognosis.

Assessment of cell line models of primary human cells by Raman spectral phenotyping.

Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures.

Secondary vascular changes in pulmonary sequestrations.

AIMS: Whilst parenchymal changes in pulmonary sequestrations are well described, there are comparatively little data on associated vascular changes and their extent. The aim of this study was to retrospectively review morphological changes within sequestrations, concentrating on vascular changes and associations with clinical parameters. METHODS AND RESULTS: Twenty-seven resected cases of sequestrations (intralobar n = 20, extralobar n = 7) showed a male predominance (n = 16) and an age range of 2 months-60 years (average 13 years). Plexogenic vascular changes (medial hypertrophy and intimal fibrosis) were seen in 15 of 27 cases, as well as plexiform lesions in seven cases. Patients with plexogenic changes had a higher mean age compared with those lacking vascular changes (19 versus 6 years) and were more commonly female. Respiratory tract infections were associated solely with intralobar sequestrations. No other associations between presenting symptoms and histopathological parameters were identified. Adjacent lung showed lesser plexogenic changes in six of 22 intralobar cases. There were features of type 2 congenital cystic adenomatoid lesions in 63% of cases. Dissection of the supplying systemic artery (n = 1), intralesional aspergilloma (n = 1) and coexistent lymphangiomatosis (n = 1) were also identified. CONCLUSIONS: Hypertensive vascular changes are not uncommon in both intrapulmonary and extrapulmonary sequestrations, although their relative severity seems unrelated to presenting symptoms.

First-line systemic treatment of advanced stage non-small-cell lung cancer in Asia: consensus statement from the Asian Oncology Summit 2009.

Non-small-cell lung cancer (NSCLC) is an increasing global challenge, especially in low-income countries. Most guidelines for the management of advanced-stage NSCLC have limited effect in countries with resource constraints. Following a systematic literature search, we present an overview of the management of advanced-stage NSCLC in the first-line setting, discuss resources required for systemic therapy, and provide treatment recommendations stratified to four resources levels. Treatment guidelines appropriate for different resource levels offer a realistic approach to management of advanced-stage NSCLC, by recognising the limitations of a particular health-care system. Although there are many barriers to cancer control in low-resource countries, these can be overcome by using measures that are culturally appropriate, economically feasible, and evidence-based. Initiatives include strategic planning, tobacco control, training of health-care workers, access to therapeutic agents, acquisition of information, public education, and alliances with established institutions and international organisations.

Lung Volume Reduction Surgery: Reinterpreted With Longitudinal Data Analyses Methodology.

BACKGROUND: The largest randomised controlled trial evaluating results of lung volume reduction surgery (LVRS) was conducted by the National Emphysema Treatment Trial (NETT) that published a series of reports for outcomes up to 24 months. However, patient outcomes were difficult to interpret due to limitations in and the presentation of conventional statistical analyses applied to longitudinal data. We reevaluated the NETT results using longitudinal data methodology to report longer-term outcomes to facilitate interpretation by clinicians and patients who are considering LVRS for emphysema management. METHODS: Trial data were released by the United States National Institutes of Health and the National Heart, Lung, and Blood Institute and analyzed using a mixed-effects model. Data on the difference in lung function variables between patients receiving LVRS vs medical care out to 5 years were estimated and are presented. RESULTS: The 5-year differences in patients randomised to LVRS were a small but sustained improvement in lung function indicators of forced expiratory volume in 1 second, forced vital capacity, and residual volume of +1.4% (P < .001), +3.44% (P < .001) and -19.49% (P < .001) of the predicted values, respectively. With regards to physiological function, the 5-year difference in patients randomised to LVRS was an overall 0.89 W improvement in maximum workload (P = .069), -4.12 improvement in shortness of breath score (P < .001), and 0.088 improvement in quality of well-being score (P = .102). CONCLUSIONS: Our results suggest that LVRS continues to have an important role in the treatment of patients with severe emphysema, with long-term benefits to lung function variables and a sustained improvement to the relief of dyspnea.

The frequency of neuroendocrine cell hyperplasia in patients with pulmonary neuroendocrine tumours and non-neuroendocrine cell carcinomas.

AIMS: To evaluate the frequency of neuroendocrine cell hyperplasia (NEH) in resected neuroendocrine tumours and non-neuroendocrine cell carcinomas and to study its relationship to selected clinical parameters. METHODS AND RESULTS: Random blocks without tumour from resected typical carcinoids (TCs, n = 46), atypical carcinoids (ACs, n = 14), large cell neuroendocrine carcinomas (LCNECs, n = 18), small cell carcinomas (SCLCs, n = 22), adenocarcinomas (ADENOs, n = 26) and squamous cell carcinomas (SCCs, n = 18) were stained for CD56 and evaluated for linear proliferations, cell aggregates (>4 CD56+ cells), and tumourlets (<5 mm with basement membrane invasion). There was a statistically significant difference between the frequency of NEH in all neuroendocrine tumours (TC/AC/LCNEC/SCLC, 35/100, 35%) (P = 0.009) when compared with non-neuroendocrine carcinomas (ADENO/SCC, 6/44, 14%) and in the frequency of NEH in TC (21/46, 46%) versus all other tumours (AC/LCNEC/SCLC/SCC/ADENO, 20/98, 20%) (P = 0.001). There was increased frequency of NEH in peripheral TCs (8/13, 62%) compared with central TCs (14/33, 43%) (P = 0.33). There was no association between smoking history and NEH. Clinical and imaging data showed no evidence of an increased frequency of obliterative bronchiolitis in patients with NEH. CONCLUSIONS: NEH is significantly increased in the background lung of neuroendocrine tumours when compared with non-neuroendocrine carcinomas, supportive data for NEH having neoplastic potential.

Updated lung cancer staging system.

The tumor, node and metastasis classification of malignant tumors is periodically revised. Its seventh edition includes the updated classification for lung cancer, based on the analyses of the International Association for the Study of Lung Cancer international database. It is the largest validation ever carried out to date: 100,869 patients registered in 46 databases from 20 countries. The analysis of this database allowed a detailed study of the impact of tumor size on prognosis, the reclassification of additional tumor nodules, the reclassification of pleural dissemination and the separation of two groups of metastatic disease. These changes led to modifications in stage grouping that better differentiate tumors with different prognosis. This updated classification is also recommended for small-cell lung cancer, and for broncho-pulmonary carcinoids. A new international nodal map with stations and zones, and a histological definition of visceral pleura invasion, have also been proposed.

Spectral monitoring of surfactant clearance during alveolar epithelial type II cell differentiation.

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids-specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.

Immortalization of human alveolar epithelial cells to investigate nanoparticle uptake.

Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 microm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 microm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-microm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cell-associated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

Physiological properties of human diaphragm muscle fibres and the effect of chronic obstructive pulmonary disease.

The contractile and actomyosin ATPase properties of single fibres were examined in human diaphragm muscle obtained from patients with and without chronic obstructive pulmonary disease (COPD). Costal diaphragm biopsies were taken from five patients without evidence of COPD and from 11 age-matched individuals with varying degrees of the disease. Our aim was to establish whether changes in contractile properties of COPD diaphragm could be fully explained by the previously documented shift towards a greater proportion of type I myosin heavy chain isoform in COPD. The relative proportion of type I diaphragm fibres from non-COPD and COPD patients was measured by gel electrophoresis, and was negatively correlated with FEV(1) over the full range of values investigated. There was also significant atrophy of the type I fibre population in COPD diaphragms. Isometric tension was similar among the fibre types and between the COPD and non-COPD patients. The intrinsic energetic properties of diaphragm fibres were examined by monitoring the time-resolved actomyosin ATPase activity in COPD and non-COPD fibres that produced similar isometric forces. The isometric ATPase rate in COPD fibres was reduced to 50% of the rate in non-COPD fibres; hence, the cost of isometric contraction in type I and type IIA COPD fibres was reduced to between one-third and one-half of the tension cost calculated for non-COPD fibres. The rate of force development in type I COPD fibres was reduced to 50% of the rate seen in non-COPD type-I fibres. No difference in the rate of ATP consumption between COPD and non-COPD fibres was evident during isovelocity shortening. These data extend previous findings showing that aspects of breathing mechanics during progressive COPD are associated with remodelling of the diaphragm fibre-type distribution; on top of the increase in type I fibres there are fibre-specific reductions in force development rate (type I fibres) and ATPase rate that are consistent with the impairment of cross-bridge cycling kinetics.

Preoperative chemotherapy in patients with resectable non-small cell lung cancer: results of the MRC LU22/NVALT 2/EORTC 08012 multicentre randomised trial and update of systematic review.

BACKGROUND: Although surgery offers the best chance of cure for patients with non-small cell lung cancer (NSCLC), the overall 5-year survival rate is modest, and improvements are urgently needed. In the 1990s, much interest was generated from two small trials that reported striking results with neo-adjuvant chemotherapy, and therefore our intergroup randomised trial was designed to investigate whether, in patients with operable non-small cell lung cancer of any stage, outcomes could be improved by giving platinum-based chemotherapy before surgery. METHODS: Patients were randomised to receive either surgery alone (S), or three cycles of platinum-based chemotherapy followed by surgery (CT-S). Before randomisation, clinicians chose the chemotherapy that would be given from a list of six standard regimens. The primary outcome measure was overall survival, which was analysed on an intention-to-treat basis. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN25582437. RESULTS: 519 patients were randomised (S: 261, CT-S: 258) from 70 centres in the UK, Netherlands, Germany, and Belgium. Most (61%) were clinical stage I, with 31% stage II, and 7% stage III. Neo-adjuvant chemotherapy was feasible (75% of patients received all three cycles of chemotherapy), resulted in a good response rate (49% [95% CI 43%-55%]) and down-staging in 31% (25%-37%) of patients, and did not alter the type or completeness of the surgery (lobectomy: S: 56%, CT-S: 60%, complete resection: S: 80%, CT-S: 82%). Post-operative complications were not increased in the CT-S group, and no impairment of quality of life was observed. However, there was no evidence of a benefit in terms of overall survival (hazard ratio [HR] 1.02, 95% CI 0.80-1.31, p=0.86). Updating the systematic review by addition of the present result suggests a 12% relative survival benefit with the addition of neoadjuvant chemotherapy (1507 patients, HR 0.88, 95% CI 0.76-1.01, p=0.07), equivalent to an absolute improvement in survival of 5% at 5 years INTERPRETATION: Although there was no evidence of a difference in overall survival with neo-adjuvant chemotherapy, the result is statistically consistent with previous trials, and therefore adds considerable weight to the current evidence.

Variation in iron homeostasis genes between patients with ARDS and healthy control subjects.

BACKGROUND: Abnormal plasma and lung iron mobilization is associated with the onset and progression of ARDS and is detectable in specific at-risk populations. Patients with ARDS also have pronounced oxidative and nitrosative stress that can be catalyzed and thereby aggravated by the bioavailability of redox active iron. ARDS of pulmonary and extrapulmonary origin may differ pathophysiologically and require different ventilatory strategies. Evidence suggests that genetic predisposition is relevant to the pathogenesis of ARDS. We therefore explored the hypothesis that polymorphisms from a panel of genes encoding iron-metabolizing proteins determine susceptibility to ARDS. METHODS: Retrospective case-control study conducted at the adult ICUs of two university hospitals. Patients with ARDS (n = 122) and healthy control subjects (n = 193) were genotyped. Sequence-specific primer polymerase chain reaction was used to genotype selected biallelic single-nucleotide polymorphisms. An audit of the patient database was conducted, and 104 of the 122 ARDS patients were eligible for the final data analysis. RESULTS: Preliminary analysis indicated differences between ARDS and healthy control subjects in the incidence of polymorphism of the gene encoding ferritin light chain. Subgroup analysis indicated the prevalence of ferritin light-chain gene -3381GG homozygotes was increased in patients with ARDS of extrapulmonary origin compared to healthy control subjects. Secondly, a common haplotype in the heme oxygenase 2 gene was reduced in patients with ARDS compared to healthy control subjects and was more evident in those with ARDS of direct or pulmonary etiology. CONCLUSIONS: These results provide preliminary evidence to suggest a distinction in the genetic background of the subpopulations studied, inferring that the ferritin light-chain gene genotype confers susceptibility to ARDS, while the heme oxygenase 2 haplotype is protective against the onset of the syndrome. Such data support further previous findings that suggest abnormalities in iron handling resulting in redox imbalance are implicated in the pathogenesis of ARDS.

Reducing cardiac injury during minimally invasive repair of pectus excavatum.

Minimally invasive repair of pectus excavatum (MIRPE) provides a minimal access approach to correct pectus excavatum deformities. Cardiovascular complications represent a rare but catastrophic complication of this cosmetic operation. We describe a modification to the technique following a case of cardiac puncture.

Mucinous cells in type 1 pulmonary congenital cystic adenomatoid malformation as mucinous bronchioloalveolar carcinoma precursors.

Type 1 congenital cystic adenomatoid malformation (CCAM), the most frequent malformation of the lung, is the only type to present intracystic mucinous cell clusters, which may form beyond the cysts extracystic mucinous proliferation resembling mucinous bronchioloalveolar carcinomas (BACs). As mucinous BACs are increasingly described in the literature in young patients with CCAM, we hypothesized that type 1 CCAM mucinous cells could represent BAC precursors. We reviewed 7 cases of type 1 CCAM including 6 with intracystic mucinous cell clusters, 3 with extracystic mucinous proliferations, and 4 with mucinous BAC or mixed adenocarcinoma with predominant BAC. K-ras mutations at codon 12 were detected in 3/3 intracystic mucinous cell clusters, in 2/3 extracystic mucinous proliferations, and in 3/4 BAC. Loss of heterozygosity (LOH) at p16(INK4) locus, with microsatellite alterations in 3 cases, was observed in 2/3 intracystic mucinous cell clusters, in 2/3 extracystic mucinous proliferations, and in all BAC. Two extracystic mucinous proliferations showed LOH at FHIT and Rb loci, respectively. P16(INK4) expression was lost in 2 intracystic mucinous cell clusters, 1 extracystic mucinous proliferation, and 1 BAC. Neither epidermal growth factor receptor mutation on exons 18, 19, and 21 nor P53 accumulation was observed. All lesions expressed MUC5AC, but were negative for MUC2, CDX2, and TTF-1. In conclusion, type 1 CCAM mucinous cells share the same differentiation profile with corresponding mucinous BAC, consistent with a common bronchial origin. Moreover, the high frequency of K-ras mutation and LOH and/or microsatellite alterations at p16(INK4) locus presented by these mucinous cells justifies their consideration as BAC precursors.

Cancer of the Esophagus and Esophagogastric Junction: An Eighth Edition Staging Primer.

This primer for eighth edition staging of esophageal and esophagogastric epithelial cancers presents separate classifications for the clinical (cTNM), pathologic (pTNM), and postneoadjuvant pathologic (ypTNM) stage groups, which are no longer shared. For pTNM, pT1 has been subcategorized as pT1a and pT1b for the subgrouping pStage I adenocarcinoma and squamous cell carcinoma. A new, simplified esophagus-specific regional lymph node map has been introduced. Undifferentiated histologic grade (G4) has been eliminated; additional analysis is required to expose histopathologic cell type. Location has been removed as a category for pT2N0M0 squamous cell cancer. The definition of the esophagogastric junction has been revised. ypTNM stage groups are identical for both histopathologic cell types, unlike those for cTNM and pTNM.

Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer.

Angiogenesis is regarded as essential for tumour growth. However, we have demonstrated that some other aggressive non-small-cell lung carcinomas (n-SCLC) do not have angiogenesis. In this study, using cDNA microarray analysis, we demonstrate that angiogenic and nonangiogenic tumour types can be distinguished by their gene expression profiles. Tissue samples from 42 n-SCLC patients were obtained with consent. In all, 12 tumours were nonangiogenic and 30 angiogenic. The two groups were matched by age, sex, smoking and tumour stage. Total RNAs were extracted followed by microarray hybridization and image scan procedure. Data were analysed using GeneSpring 5.1 software. A total of 62 genes were found to be able to separate angiogenic from nonangiogenic tumours. Nonangiogenic tumours have higher levels of genes concerned with mitochondrial metabolism, mRNA transcription, protein synthesis and the cell cycle. Angiogenic tumours have higher levels of genes coding for membrane vesicles, integrins, remodelling, angiogenesis and apoptosis. These results further support our first finding that nonangiogenic lung tumours are fast-growing tumours filling the alveoli in the absence of vascular remodelling. We raise the hypothesis that in nonangiogenic tumours, hypoxia leads to a higher activation of the mitochondrial respiratory chain, which allows tumour growth without triggering angiogenesis.

Recurrent KRAS codon 146 mutations in human colorectal cancer.

An activating point mutation in codon 12 of the HRAS gene was the first somatic point mutation identified in a human cancer and established the role of somatic mutations as the common driver of oncogenesis. Since then, there have been over 11,000 mutations in the three RAS (HRAS, KRAS and NRAS) genes in codons 12, 13 and 61 reported in the literature. We report here the identification of recurrent somatic missense mutations at alanine 146, a highly conserved residue in the guanine nucleotide binding domain. In two independent series of colorectal cancers from Hong Kong and the United States we detected KRAS A146 mutations in 7/126 and 2/94 cases, respectively, giving a combined frequency of 4%. We also detected KRAS A146 mutations in 2/40 (5%) colorectal cell lines, including the NCI-60 colorectal cancer line HCC2998. Codon 146 mutations thus are likely to make an equal or greater contribution to colorectal cancer than codon 61 mutations (4.2% in our combined series, 1% in the literature). Lung adenocarcinomas and large cell carcinomas did not show codon 146 mutations. We did, however, identify a KRAS A146 mutation in the ML-2 acute myeloid leukemia cell line and an NRAS A146 mutation in the NALM-6 B-cell acute lymphoblastic leukemia line, suggesting that the contribution of codon 146 mutations is not entirely restricted to colorectal cancers or to KRAS.

Passive properties of the diaphragm in COPD.

Structural adaptations that occur in the diaphragm muscle of patients with chronic obstructive pulmonary disease (COPD), namely an increase in type I fibers and a decrease in type II fibers, have been explored in terms of the active contractile properties of the diaphragm. The aim of this study was to test the passive properties of the diaphragm by measuring the force response of relaxed diaphragm muscle fibers to stretching to determine the effect of COPD on these properties. Costal diaphragm biopsies were taken from patients with COPD and from controls with normal pulmonary function. From these biopsies, titin expression was assessed in diaphragm homogenates by gel electrophoresis, and the restoring force was measured by incremental stretching of single fibers in the relaxed state and measuring the force response to stretching. A quadratic model was used to illustrate the relationship between restoring force and muscle fiber length, and it revealed that COPD fibers generate significantly lower restoring forces than control fibers as judged by the area under the force-length curve. Furthermore, this finding applies to both type I and type II fibers. Gel electrophoresis revealed different titin isoforms in COPD and controls, consistent with the conclusion that COPD results not only in a change in muscle fiber-type distribution but in a structural change in the titin molecule in all muscle fiber types within the diaphragm. This may assist the muscle with the energetic changes in the length of the diaphragm required during breathing in COPD.