David C.L. Lam

Molecular biology of lung cancer.

Lung cancers are characterised by abundant genetic diversity with relatively few recurrent mutations occurring at high frequency. However, the genetic alterations often affect a common group of oncogenic signalling pathways. There have been vast improvements in our understanding of the molecular biology that underpins lung cancer in recent years and this has led to a revolution in the diagnosis and treatment of lung adenocarcinomas (ADC) based on the genotype of an individuals tumour. New technologies are identifying key and potentially targetable genetic aberrations not only in adenocarcinoma but also in squamous cell carcinoma (SCC) of the lung. Lung cancer mutations have been identified in v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), BRAF and the parallel phosphatidylinositol 3-kinase (PI3K) pathway oncogenes and more recently in MEK and HER2 while structural rearrangements in ALK, ROS1 and possibly rearranged during transfection (RET) provide new therapeutic targets. Amplification is another mechanism of activation of oncogenes such as MET in adenocarcinoma, fibroblastgrowth factor receptor 1 (FGFR1) and discoidin domain receptor 2 (DDR2) in SCC. Intriguingly, many of these genetic alternations are associated with smoking status and with particular racial and gender differences, which may provide insight into the mechanisms of carcinogenesis and role of host factors in lung cancer development and progression. The role of tumour suppressor genes is increasingly recognised with aberrations reported in TP53, PTEN, RB1, LKB11 and p16/CDKN2A. Identification of biologically significant genetic alterations in lung cancer that lead to activation of oncogenes and inactivation of tumour suppressor genes has the potential to provide further therapeutic opportunities. It is hoped that these discoveries may make a major contribution to improving outcome for patients with this poor prognosis disease.

Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non–Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChR) on bronchial epithelial cells, can regulate cellular proliferation and apoptosis via activating the Akt pathway. Delineation of nAChR subtypes in non-small-cell lung cancers (NSCLC) may provide information for prevention or therapeutic targeting. Expression of nAChR subunit genes in 66 resected primary NSCLCs, 7 histologically non-involved lung tissues, 13 NSCLC cell lines, and 6 human bronchial epithelial cell lines (HBEC) was analyzed with quantitative PCR and microarray analysis. Five nonmalignant HBECs were exposed to nicotine in vitro to study the variation of nAChR subunit gene expression with nicotine exposure and removal. NSCLCs from nonsmokers showed higher expression of nAChR alpha6 (P < 0.001) and beta3 (P = 0.007) subunit genes than those from smokers, adjusted for gender. In addition, nAChR alpha4 (P < 0.001) and beta4 (P = 0.029) subunit gene expression showed significant difference between NSCLCs and normal lung. Using Affymetrix GeneChip U133 Sets, 65 differentially expressed genes associated with NSCLC nonsmoking nAChR alpha6beta3 phenotype were identified, which gave high sensitivity and specificity of prediction. nAChR alpha1, alpha5, and alpha7 showed significant reversible changes in expression levels in HBECs upon nicotine exposure. We conclude that between NSCLCs from smokers and nonsmokers, different nAChR subunit gene expression patterns were found, and a 65-gene expression signature was associated with nonsmoking nAChR alpha6beta3 expression. Finally, nicotine exposure in HBECs resulted in reversible differences in nAChR subunit gene expression. These results further implicate nicotine in bronchial carcinogenesis and suggest targeting nAChRs for prevention and therapy in lung cancer.

Randomised study of three non‐surgical treatments in mild to moderate obstructive sleep apnoea

Background: Patients with mild to moderate obstructive sleep apnoea (OSA) may be managed with different treatment options. This study compared the effectiveness of three commonly used non-surgical treatment modalities. Methods: Subjects with mild to moderate OSA were randomised to one of three treatment groups for 10 weeks: conservative measures (sleep hygiene) only, continuous positive airways pressure (CPAP) in addition to conservative measures or an oral appliance in addition to conservative measures. All overweight subjects were referred to a weight-reduction class. OSA was assessed by polysomnography. Blood pressure was recorded in the morning and evening in the sleep laboratory. Daytime sleepiness was assessed with the Epworth Sleepiness Scale. Health-related quality of life (HRQOL) was assessed with the 36-Item Short-Form Health Survey (SF-36) and Sleep Apnoea Quality of Life Index (SAQLI). Results: 101 subjects with a mean (SEM) apnoea–hypopnoea index (AHI) of 21.4 (1.1) were randomised to one of the three groups. The severity of sleep-disordered breathing was decreased in the CPAP and oral appliance groups compared with the conservative measures group, and the CPAP group was significantly better than the oral appliance group. Relief from sleepiness was significantly better in the CPAP group. CPAP was also better than the oral appliance or conservative measures in improving the “bodily pain” domain, and better than conservative measures in improving the “physical function” domain of SF-36. Both CPAP and the oral appliance were more effective than conservative measures in improving the SAQLI, although no difference was detected between the CPAP and oral appliance groups. CPAP and the oral appliance significantly lowered the morning diastolic blood pressure compared with baseline values, but there was no difference in the changes in blood pressure between the groups. There was also a linear relationship between the changes in AHI and body weight. Conclusion: CPAP produced the best improvement in terms of physiological, symptomatic and HRQOL measures, while the oral appliance was slightly less effective. Weight loss, if achieved, resulted in an improvement in sleep parameters, but weight control alone was not uniformly effective.

C-Reactive Protein Is Associated With Obstructive Sleep Apnea Independent of Visceral Obesity

BACKGROUND Obstructive sleep apnea (OSA) is associated with adverse cardiovascular outcomes. C-reactive protein (CRP) predicts atherosclerotic complications. Our study evaluates whether OSA is associated with an elevated CRP level, after elimination of known confounders including visceral obesity. METHODS Men without significant chronic medical illness, regular medications, or illness in the preceding 4 weeks were enrolled. Subjects with morbid obesity, newly detected high BP, or fasting glucose were excluded. They underwent polysomnography and MRI of abdomen to quantify visceral fat volume. High-sensitivity CRP levels were measured. RESULTS 111 men with mean body mass index (BMI) 26.3 +/- 3.8 kg/m(2) were evaluated. After adjustment for age, smoking, BMI, waist circumference, and sleep efficiency, CRP correlated positively with the apnea-hypopnea index (AHI) [r = 0.35, p < 0.001], duration of O(2) saturation < 90% (r = 0.29, p = 0.002), and arousal index (r = 0.32, p = 0.001), and it correlated negatively with minimal O(2) saturation (r = -0.29, p = 0.002). These correlations were consistent when adjustment was made for MRI visceral fat volume instead of waist circumference. In the regression model, significant predictors of CRP included AHI, waist circumference, and triglycerides (adjusted R(2), 0.33, p = 0.001, p = 0.002, p = 0.018, respectively). Among the 111 subjects, 32 subjects with no or mild OSA (AHI < 15 events/h) were matched with 32 subjects with moderate-to-severe OSA (AHI > or = 15 events/h) in MRI visceral fat volume. CRP was higher in subjects with moderate-to-severe OSA (median, 1.32; 0.45 to 2.34 mg/L) when compared to subjects with no or mild OSA (median, 0.54; 0.25 to 0.89 mg/L; p = 0.001). CONCLUSIONS In healthy middle-aged men, elevated CRP level is associated with OSA independent of visceral obesity.

Electronic Cigarettes A Position Statement of the Forum of International Respiratory Societies

BACKGROUND Awareness and usage of electronic cigarettes has exponentially increased during the last few years, especially among young people and women in some countries. The rapid acceptance of electronic cigarettes may be attributed in part to the perception created by marketing and the popular press that they are safer than combustible cigarettes. GOALS To alert and advise policy makers about electronic cigarettes and their potential hazards. METHODS Using The Unions position paper on electronic cigarettes as the starting template, the document was written using an iterative process. Portions of the manuscript have been taken directly from the position papers of participating societies. RESULTS Because electronic cigarettes generate less tar and carcinogens than combustible cigarettes, use of electronic cigarettes may reduce disease caused by those components. However, the health risks of electronic cigarettes have not been adequately studied. Studies looking at whether electronic cigarettes can aid smoking cessation have had inconsistent results. Moreover, the availability of electronic cigarettes may have an overall adverse health impact by increasing initiation and reducing cessation of combustible nicotine delivery products. CONCLUSIONS The health and safety claims regarding electronic nicotine delivery devices should be subject to evidentiary review. The potential benefits of electronic cigarettes to an individual smoker should be weighed against potential harm to the population of increased social acceptability of smoking and use of nicotine, the latter of which has addictive power and untoward effects. As a precaution, electronic nicotine delivery devices should be restricted or banned until more information about their safety is available. If they are allowed, they should be closely regulated as medicines or tobacco products.

Prevalence and recognition of obstructive sleep apnea in Chinese patients with type 2 diabetes mellitus.

BACKGROUND Obstructive sleep apnea (OSA) is associated with disorders of glucose metabolism. Previous studies revealed a high prevalence of OSA among subjects with type 2 diabetes mellitus (DM). The aims of this study were to determine the prevalence of OSA and associated clinical factors in Chinese patients with DM. METHODS All records of the DM clinic at a teaching hospital in Hong Kong were screened between January 2007 and June 2008. Inclusion criteria for patients were Chinese, aged 18 to 75 years, with type 2 DM. Patients with unstable medical illnesses, gestational diabetes, or on renal replacement therapy were excluded. RESULTS Of 3,489 records screened, 1,859 subjects were eligible. A random sample of 663 (mean age, 58.2 ± 10.8; mean BMI, 26.0 ± 4.6), except six with known OSA, were invited for polysomnography (PSG). Of 165 subjects on which PSG was performed, OSA was diagnosed (apnea-hypopnea index [AHI] ≥ 5.0/h) in 89 subjects (53.9%, median Epworth Sleepiness Scale, 6 [interquartile range 3, 10]). Fifty-four (32.7%) had moderate/severe OSA (AHI ≥ 15/h). The estimated OSA prevalence in this diabetic cohort was 17.5% (24.7% in men, 10.3% in women). Regression analysis identified that AHI was associated independently with higher BMI, advanced age, male sex, and higher diastolic BP (R(2) = 29.6%). The adjusted OR of requiring three or more antihypertensive drugs in moderate/severe OSA was 2.48 (95% CI, 1.05-5.87). No association between glycemic control (HbA1c) and sleep was identified. CONCLUSIONS In conclusion, OSA is more prevalent in Chinese adults with DM than in the general population. A high index of suspicion for OSA in patients with DM is warranted, because they may not have overt daytime sleepiness.

EGFR Array: Uses in the Detection of Plasma EGFR Mutations in Non–Small Cell Lung Cancer Patients

Introduction: We aim to develop a simple and sensitive array-based method for the detection of epidermal growth factor receptor (EGFR) gene mutations in the plasma of non–small-cell lung cancer patients and determine its use in the follow-up of those on tyrosine-kinase inhibitor (TKI) therapy. Method: DNA from 100 &mgr;l of plasma was amplified in the presence of peptide nucleic acid clamp to provide single-stranded template for the allele-specific arrayed primer extension reaction, incorporating cyanine-5-deoxycytidine triphosphate in the newly synthesized strands. The fluorescent product was visualized by laser at 670 nm. Results: Eleven different types of EGFR TKI drug-sensitive mutants (SM) were identified in plasma-DNA from 46 of 51 patients. Five patients carried only wild-type sequence. Plasma-DNA finding was concordant in 36 of 37 cases with tumor-sequencing data. This method could detect as little as 62.5 copies of mutant L858R; 125 copies of E709K + G719A or 625 copies of del 746–750 in the presence of 100,000 copies of wild-type EGFR. In 21 patients on longitudinal follow-up for up to 18 months, SM was found in all initial plasma samples, except for three samples collected after recent chemotherapy. Nine of 16 patients (56%) who responded to TKI had undetectable plasma EGFR mutant. SM was present concurrently with drug-resistant mutant in 44% of patients with disease progression while on TKI, the remaining 56% might have other mechanisms of resistance. Conclusion: The EGFR array provides a sensitive, inexpensive, and robust method for monitoring non–small-cell lung cancer patients’ response to TKI, and obviates the need of repeated lung biopsy.

Sputum cytology examination followed by autofluorescence bronchoscopy: A practical way of identifying early stage lung cancer in central airway

BACKGROUND The prognosis of early stage lung cancer was superior to that of late stages. We hypothesize that by using sputum cytology as the first screening method followed by autofluorescence bronchoscopy could detect early stage lung cancer in the central airway. METHODS During 18-month recruitment period, subjects at high risk for lung cancer (ever smoker accumulated more than 20 pack-year and above 40 years) followed up at Chest Clinics were invited to submit sputum for cytological examination. Subjects with sputum atypia were invited to have bronchoscopy, and CT thorax. After a mean follow-up of 39+/-14 months, the characteristics of lung cancers detected in the group with sputum atypia and the group with normal sputum at baseline were assessed. RESULTS 181 subjects submitted sputum and primary lung cancer were diagnosed in 13. 46.2% of the lung cancers were in early stages. Bronchoscopy were performed in 85, and seven were confirmed to have lung cancer (six were in early stages). 81 had CT done and 92.6% had radiological abnormalities, though three lung cancers (all stage 0) were missed by CT. Five more primary lung cancers were diagnosed during the follow-up period: one in sputum atypia group and the other four (three were advanced adenocarcinoma) in normal sputum group. The overall sensitivity of sputum cytology in detecting lung cancer was 71.4% for all histology and 100% for squamous cell lung cancer. CONCLUSIONS Sputum cytology examination followed by bronchoscopy was a practical way of detecting early stage lung cancer in central airway.

Establishment and Expression Profiling of New Lung Cancer Cell Lines from Chinese Smokers and Lifetime Never-Smokers

Background: Bronchogenic adenocarcinoma is the predominant histologic subtype of lung cancer, which ranks top in the cancer mortality in both men and women. Female nonsmokers and adenocarcinoma have emerged as a distinct combination in patients with lung cancer in recent decades. Lung cancer cell lines established from patients with known clinical characteristics such as gender and smoking habit would be useful for future research on lung cancer. Methods: Four new lung adenocarcinoma cell lines (HKULC 1–4) were established from Chinese patients with primary lung adenocarcinomas and with different clinical characteristics with respect to age, gender, smoking habits, tumor staging, and previous therapy. They were characterized by immunohistochemical and growth kinetic studies, tests for tumorigenicity in nude mice, epidermal growth factor receptor (EGFR) gene mutation analysis, and in situ hybridization, and gene expression profiling with Affymetrix GeneChip HG-U133A. Results: The newly established HKULC lung adenocarcinoma cell lines were maintained for over 70 passages and demonstrated morphologic and immunohistochemical features and growth kinetics of tumor cell lines. One of the four HKULC cell lines, HKULC 3 (derived from a female nonsmoking patient with lung adenocarcinoma), was found to have a deletion at exon 19 of the EGFR gene. EGFR in situ hybridization showed no EGFR gene amplification in these cell lines. HKULC 1 and 4 formed tumor xenografts after inoculation in nude mice. A list of 71 genes that were differentially expressed or showing class predictive significance was identified. These genes included putative tumor suppressor genes (DKK3, SERPINF1, CDH11, DSC3, and KLF6), genes involved in or related to the EGFR pathways (ERBB3, MUC1, VAV1), genes involved in regulation of cell cycle and proliferation (CDKN1A and CDKN2A), a putative oncogene (EEF1A2), and a gene related to metastasis (MTSS1). Discussion: Four new lung adenocarcinoma cell lines were established from patients with different clinical characteristics. These characterized cell lines and their gene expression profiles will provide resources for studies of lung cancer biology and in vitro chemotherapeutic drug study.

Increased serum levels of advanced glycation end-products is associated with severity of sleep disordered breathing but not insulin sensitivity in non-diabetic men with obstructive sleep apnoea

BACKGROUND Patients with diabetes mellitus are known to have increased serum levels of advanced glycation end-products (AGEs), and this is also associated with insulin resistance. This study aimed to investigate the relationship between serum AGEs and insulin sensitivity in non-diabetic subjects with obstructive sleep apnoea (OSA). METHODS Adult males with no known comorbidities were recruited from the sleep clinic of a university teaching hospital. They underwent overnight in-laboratory polysomnography. Fasting blood was taken to measure serum AGE and plasma glucose levels. Insulin sensitivity was estimated using the short insulin tolerance test. RESULTS In total, 105 subjects with a mean age of 43.5 (standard deviation [SD] 9.2)years, mean body mass index of 27.1 (SD 4.0)kg/m(2), and median apnoea-hypopnoea index (AHI) of 17 (interquartile range 5-46) were analysed. Serum AGE levels were significantly higher in subjects with OSA (AHI ≥5), compared with those without OSA (AHI <5) (3.9 [SD 1.2] vs. 3.2 [SD 0.8]μg/ml, respectively; P=0.037) after adjusting for confounders. AGE levels were positively correlated with AHI (r=0.318, P=0.001), but not with insulin sensitivity. AGE levels decreased in subjects with moderate-to-severe OSA who received continuous positive airway pressure (CPAP) treatment for three months (n=18, P=0.017). CONCLUSIONS Serum AGE levels correlate with AHI in non-diabetic adult males. This relationship cannot be explained by insulin sensitivity. Supporting the hypothesis of a direct relationship between AHI and AGEs, AGE levels were found to decline with CPAP therapy.