Methylation analysis in spontaneous sputum for lung cancer diagnosis

Objectives

Lung cancer is the most fatal cancer in the developed world due to presence of metastases at time of diagnosis. The aim of this study is to examine DNA hypermethylation in sputum compared to sputum cytology for the diagnosis of lung cancer. A novel risk analysis is introduced, using the distinction between diagnostic and risk markers.

Methods

Two independent sets were randomly composed from a prospectively collected sputum bank (Set 1: n = 98 lung cancer patients, n = 90 controls; Set 2: n = 60 lung cancer patients, n = 445 controls). Sputum cytology was performed for all samples. The following DNA hypermethylation markers were tested in both sets: RASSF1A, APC and cytoglobin (CYGB). Two statistical analyses were conducted: multivariate logistic regression and a risk classification model based on post-test probabilities.

Results

In multivariate analysis, RASSF1A was the best of the three markers in discriminating lung cancer cases from controls in both sets (sensitivity 41–52%, specificity 94–96%). The risk model showed that 36% of lung cancer patients were defined as “high risk” (≥60% chance on lung cancer) based on RASSF1A hypermethylation in Set 1. The model was reproducible in Set 2. Risk markers (APC, CYGB) have less diagnostic value.Sensitivity of cytology for lung cancer diagnosis was 22%. RASSF1A hypermethylation yielded a sensitivity of 45%. The combined sensitivity for RASSF1A with cytological diagnosis increased to 52% with similar specificity (94%).

Conclusion

In a diagnostic setting, hypermethylation analysis in sputum is possible when a diagnostic marker is used. However, risk markers are insufficient for this purpose.     

Performance evaluation of the DNA methylation biomarker SHOX2 for the aid in diagnosis of lung cancer based on the analysis of bronchial aspirates

In the identification of subjects with lung cancer, increased DNA methylation of the SHOX2 gene locus in bronchial aspirates has previously been proven to be a clinically valuable biomarker. This is particularly true in cases where the cytological and histological results following bronchoscopy are undetermined. This previous case control study was conducted using research assay components and a complex work flow. To facilitate the use in a diagnostic setting, a CE marked in?vitro diagnostic test kit to quantify SHOX2 DNA methylation in bronchial aspirates was developed and characterized. The presented assay for measuring SHOX2 DNA methylation in bronchial aspirates is based on two major steps: generation of bisulfite converted template DNA from patient samples followed by subsequent determination of SHOX2 biomarker methylation by real-time PCR. Individual kits for DNA preparation, real-time PCR analysis and work flow control were developed. This study describes the analytical performance (reproducibility, accuracy, interfering substances, cross-reactivity) of the in vitro diagnostic (IVD) test kit 'Epi proLung BL Reflex Assay'. In addition, the intended use of the test was validated in a clinical performance evaluation (case control) study comprised of 250 patients (125?cases, 125?controls). The results describe the test as a robust and reliable diagnostic tool for identifying patients with lung cancer using Saccomanno-fixed bronchial lavage specimens (AUC [95% confidence intervals] = 0.94 [0.91-0.98], sensitivity 78% [69-86]/specificity 96% [90-99]). This test may be used as a diagnostic adjunct to existing clinical and pathological investigations in lung cancer.     

肺癌侵袭近端支气管壁的临床病理探讨

目的探讨不同组织类型肺癌向近端支气管壁侵袭的规律。方法对１５１例中心型肺癌手术切除标本近端支气管进行不同断面的病理学研究。其中,全肺切除标本４１例,肺叶切除标本１１０例。结果癌细胞沿管壁粘膜下层或多层面直接侵袭是癌扩延的主要方式,９６．６％的癌侵袭发生在距瘤缘１．５ｃｍ以内的管壁上。其侵袭距离与病理类型、侵袭方式及ＴＮＭ分期因子（ｐＴ、ｐＮ）相关。转移淋巴结对管壁的侵袭也是癌扩延不容忽视的形式。结论为获得根治切除,支气管切端距瘤缘的安全界限原则上应超过１．５ｃｍ,并须彻底清除肺门纵隔淋巴结     

Puncture of a bronchial tumor in the bronchoscopic diagnosis of lung cancer

An analysis of the results of bronchial tumor biopsy performed in 200 lung cancer patients is reported. Morphological verification proved feasible in 193 of 200 patients. Biopsy and puncturing of the tumor are found to be most advantageous technics for the material take during bronchoscopy. Contrary to biopsy, an efficiency of transbronchial puncturing is not affected by the character of tumor growth and its morphological structure. A combined use of biopsy and puncture makes it possible to increase the percentage of morphologically supported diagnosis of pulmonary cancer. Transbronchial puncturing is also an efficient maneuver for detection of cancer recurrence in the bronchial stump and a residual tumor after conservative therapy. Endoscopic signs of the peribronchial pattern of tumor growth are absolute indications to its puncture.     

Pulmonary tuberculosis increases the risk of lung cancer: a population-based cohort study

BACKGROUND:

The possible effect of pulmonary tuberculosis (TB) on subsequent lung cancer development has been suspected, but the evidence remains inconsistent. The purpose of this study was to perform a nationwide population‐based cohort study to investigate the risk of lung cancer after pulmonary TB infection.

METHODS:

This nationwide population‐based cohort study was based on data obtained from the Taiwan National Health Insurance Database. In total, 5657 TB patients and 23,984 controls matched for age and sex were recruited for the study from 1997 to 2008.

RESULTS:

The incidence rate of lung cancer (269 of 100,000 person‐years) was significantly higher in the pulmonary TB patients than that in controls (153 of 100,000 person‐years) (incidence rate ratio [IRR], 1.76; 95% confidence interval [CI], 1.33‐2.32; P < .001). Compared with the controls, the IRRs of lung cancer in the TB cohort were 1.98 at 2 to 4 years, 1.42 at 5 to 7 years, and 1.59 at 8 to 12 years after TB infections. The multivariate Cox proportional hazards model revealed pulmonary TB infections (hazard ratio [HR], 1.64; 95% CI, 1.24‐2.15; P < .001) and chronic obstructive pulmonary disease (HR, 1.09; 95% CI, 1.03‐1.14; P = .002) to be independent risk factors for lung cancer.

CONCLUSIONS:

Pulmonary infection with TB is associated with an increased risk of lung cancer. Cancer 2011. © 2010 American Cancer Society.     

Proximal bronchial invasion of lung cancer: A clinicopathological study

FromJanuary1994toDecember1996,151operativelyresectedspecimensfromhilarlungcancercasesorpatientsweresubjecttohistopathologicalstudyinHenanCancerHospital.Thepurposewastoexplorepathologicallythecharacteristicsofproximalbronchialinvasionoflungcancerwithvarioushistopathologictypes,andthentoprovidetherationalbasisforthedeterminationofsaferesectionsizeofbronchusandtheselectionofaproperoperationmode.MATERIALSANDMETHODSClinicalDataThe151specimenswereallnewlyresectedduringpneumonectomyfrompati…     

Magnetic enrichment of bronchial epithelial cells from sputum for lung cancer diagnosis

BACKGROUND: Sputum is an easily accessible diagnostic material for lung cancer early detection by cytologic and molecular genetic analysis of exfoliated airway epithelial cells. However, the use of sputum is limited by its cellular heterogeneity, which includes >95% macrophages and neutrophils and only about 1% bronchial epithelial cells. We propose to obtain concentrated and purified bronchial epithelial cells to improve early detection of lung cancer in sputum samples. METHODS: Sputum was collected from patients with stage I nonsmall-cell lung cancer, cancer-free smokers, and healthy nonsmokers. Magnetic-assisted cell sorting (MACS) with anti-CD14 and anti-CD16 antibody beads were used to enrich bronchial epithelial cells by depleting macrophages and neutrophils from sputum. Fluorescence in situ hybridization (FISH) analysis for detection of FHIT deletion and cytology were evaluated in the enriched specimens. RESULTS: The bronchial epithelial cells were concentrated to 40% purity from 1.1% of the starting population, yielding an average of 36-fold enrichment and at least 2.3 x 10(5) cells per sample. Detecting FHIT deletions for lung cancer diagnosis produced 58% sensitivity in the enriched sputum, whereas there was 42% sensitivity in the unenriched samples (P = .02). Cytologic examination of the enriched sputum resulted in 53% sensitivity, as compared with 39% sensitivity in unenriched sputum (P = .03). Furthermore, only 2 cytocentrifuge slides of the unenriched sputum were needed for the analyses, as compared with up to 10 cytocentrifuge slides required from the unprocessed specimens. CONCLUSIONS: The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer.     

Quantitative analysis of a novel DNA hypermethylation panel using bronchial specimen for lung cancer diagnosis

Non-diagnostic findings are common in transbronchial lung biopsy (TBLB) and endobronchial ultrasound-guided transbronchial lung biopsy (EBUS-TBLB). One of the challenges is to improve the detection of lung cancer using these techniques. To address this issue, we utilized an 850 K methylation chip to identify methylation sites that distinguish malignant from benign lung nodules. Our study found that a combination of HOXA7, SHOX2 and SCT methylation analysis has the best diagnostic yield in bronchial washing (sensitivity: 74.1%; AUC: 0.851) and brushing samples (sensitivity: 86.1%; AUC: 0.915). We developed a kit comprising these three genes and validated it in 329 unique bronchial washing samples, 397 unique brushing samples and 179 unique patients with both washing and brushing samples. The panel's accuracy in lung cancer diagnosis was 86.9%, 91.2% and 95% in bronchial washing, brushing and washing + brushing samples, respectively. When combined with cytology, rapid on-site evaluation (ROSE), and histology, the panel's sensitivity in lung cancer diagnosis was 90.8% and 95.8% in bronchial washing and brushing samples, respectively, and 100% in washing + brushing samples. Our findings suggest that quantitative analysis of the three-gene panel can improve the diagnosis of lung cancer using bronchoscopy.     

Smoking cessation among men following cancer diagnosis: a matched cohort study

Purpose

Cigarette smoking among cancer survivors increases the risk of recurrence and secondary cancers. We sought to investigate smoking cessation following diagnosis of cancer compared to those not diagnosed with cancer. We also investigated cessation following diagnosis of a smoking-related and non-smoking-related cancer separately.

Methods

We conducted a matched cohort study within the Health Professionals Follow-Up Study (HPFS). We identified 566 men diagnosed with cancer who were current cigarette smokers at the time of diagnosis between 1986 and 2010 (exposed). Men diagnosed with cancer were age-matched 1:4 to men without a diagnosis of cancer who were also current cigarette smokers (unexposed). Multivariable conditional logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (CI) to evaluate the association between a cancer diagnosis and smoking cessation within 2 and 4 years post diagnosis adjusted for potential confounders, overall and for smoking-related and non-smoking-related cancers.

Results

Of the men with cancer, 38% quit within 2 years and 42% within 4 years of diagnosis. Men diagnosed with cancer were more likely to quit smoking within 2 (OR?=?2.5, 95% CI: 2.0–3.0) and 4 years (OR?=?1.6, 95% CI: 1.3–2.0) post diagnosis, compared to matched men without cancer. The association was similar for smoking-related (OR?=?3.4, 95%: 1.6–7.2) and non-smoking-related cancers (OR?=?3.8, 95%: 2.8–5.2).

Conclusions

Men diagnosed with cancer were more likely to quit smoking compared to men not diagnosed with cancer. A cancer diagnosis may be a “teachable moment” in which strategies to promote smoking cessation for individuals diagnosed with smoking-related and non-smoking-related cancers should be investigated.

Implications for Cancer Survivors

There is a continued need for the widespread implementation of cessation interventions for cancer survivors.

     

Methylation profiling of archived non-small cell lung cancer: a promising prognostic system.

PURPOSE: Enhanced prognostication power is becoming more desirable in clinical oncology. In this study, we explored the prognostic potential of multigene hypermethylation profiling in non-small-cell lung cancer. EXPERIMENTAL DESIGN: We evaluated a panel of eight genes (p16, APC, ATM, hMLH1, MGMT, DAPK, ECAD, and RASSF1A) using methylation-specific PCR in 105 archived specimens of non-small-cell lung cancer representing all stages of the illness. We analyzed the effect of gene methylation status on outcome individually in a cumulative manner and in a combinatorial approach using recursive partitioning to identify methylation profiles, which affect overall survival. RESULTS: In this data set, tumors harboring promoter hypermethylation at two or more genes exhibit similar survival trends to others in the cohort. Using recursive partitioning, three genes (APC, ATM, and RASSF1A) emerged as determinants of prognostic groups. This designation retained its statistical significance even when disease stage and age were entered into a multivariate analysis. Using this approach, patients whose tumors were hypermethylated at APC and those hypermethylated at only ATM (not also at APC or RASSF1A) enjoyed substantially longer 1- and 2-year survival than patients in the remaining groups. In 32 adjacent histologically normal lung tissue specimens, we detected similar methylation abnormalities. CONCLUSION: Assessment of promoter hypermethylation aberrations may facilitate prognostic profiling of lung tumors, but validation in independent data sets is needed to verify these profiles. This system uses material that is abundantly available with linked outcome data and can be used to generate reliable epigenetic determinants.     

支气管刷检液基细胞学在肺癌诊断中的应用价值

目的:探讨支气管刷检液基细胞学在肺癌诊断中的应用价值。方法:支气管刷检标本做液基细胞学检测,剩余标本制作成细胞块切片。结果:液基细胞学诊断肺癌的敏感度、特异度、准确度分别为89.5%、94.3%、91.4%,分型诊断准确率为90.4%。细胞块切片诊断肺癌的敏感度、特异度、准确度分别为95.1%、100%、97.4%,分型诊断准确率为84.6%。两组的敏感度、特异度、准确度及分型诊断准确率比较差异均无统计学意义（P>0.05）。结论:液基细胞学诊断肺癌的敏感度、特异度高,大部分肺癌能准确分型,与细胞块切片合用有互补作用,液基细胞学可作为肺癌早期诊断的有效方法。     

Aberrant methylation of the adenomatous polyposis coli promoter 1A in bronchial aspirates from patients with suspected lung cancer

Promoter hypermethylation is a major mechanism for gene silencing and offers a promising starting point for developing molecular biomarkers. The purpose of our study was to determine aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A with respect to its prevalence and quantitative level in bronchial aspirates from patients with suspected lung cancer. Applying quantitative methylation-specific PCR, 155 bronchial aspirates from patients with non-small cell cancer (NSCLC) and small cell cancer (SCLC) of the lung as well as 67 bronchial aspirates from patients diagnosed for nonneoplastic lung disease were examined in a retrospective case-control study. Aberrant APC promoter 1A methylation was seen in 71% of NSCLCs, 38% of SCLCs and 42% of patients with nonneoplastic lung disease, being therefore not specific for the presence of primary lung cancer. In contrast, quantitative analysis showed a significantly higher methylation level of bronchial aspirates from NSCLC as compared to patients without neoplastic lung disease. Introducing a cutoff point that defined high level of APC hypermethylation NSCLC could be discriminated from cases without neoplastic disease with a specificity of 98.5% and a sensitivity of 39%. The data suggest that quantitative analysis of APC hypermethylation may serve as a biomarker of primary lung cancer.     

Cholecystectomy and the incidence of breast cancer: a cohort study.

A cohort comprising 11678 women who had undergone cholecystectomy in the period 1964 through 1967 for a benign gallbladder disease was investigated. They represented almost a total ascertainment from a defined geographic area. Follow-up during 11-14 completed years of observation revealed a total of 202 breast cancer cases after the cholecystectomy. This number was close to the expected incidence of 199.1 (relative risk 1.0). Further analysis of the risk in relation to duration of observation and age at operation did not reveal any trend or subgroup with a significantly increased or decreased risk. It was concluded that despite the many epidemiologic observations indicating that in Western Countries dietary habits are particularly important determinants of the high incidence of both gallstone disease and breast cancer, our results contradict the idea that the diseases share common aetiologic factors.     

Analysis of the MammaPrint breast cancer assay in a predominantly postmenopausal cohort.

PURPOSE: Most node-negative breast cancer patients are older and postmenopausal and are increasingly being offered adjuvant chemotherapy despite their low overall risk of distant relapse. A molecular diagnostic test with high negative predictive value (NPV) for distant metastasis in this subgroup would spare many older breast cancer patients adjuvant treatment. EXPERIMENTAL DESIGN: We determined the NPV and positive predictive value of the MammaPrint assay in breast cancer patients who were consecutively diagnosed and treated at the Massachusetts General Hospital between 1985 and 1997. Primary tumors from 100 patients with node-negative, invasive breast cancer (median age, 62.5 years; median follow-up, 11.3 years) were subjected to MammaPrint analysis and classified as being at either low or high risk for distant metastasis. RESULTS: The MammaPrint 70-gene signature displayed excellent NPV as in previous studies, correctly identifying 100% of women at low risk for distant metastases at 5 years. However, this assay had a lower positive predictive value (12% at 5 years) than previously observed. CONCLUSIONS: The MammaPrint assay was originally designed to identify younger breast cancer patients at low risk for distant metastasis, who might consequently be spared systemic treatment. We show here that the same signature has a very high NPV for distant recurrence after adjuvant treatment in older breast cancer patients.