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Ann Thorac Surg 2005;80:1241-1247
© 2005 The Society of Thoracic Surgeons

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Original article: General thoracic

Can We Improve the Cytologic Examination of Malignant Pleural Effusions Using Molecular Analysis?

^a Department of Surgery, Division of Thoracic Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland
^b Department of Medical Oncology, The Johns Hopkins Medical Institutions, Baltimore, Maryland
^c Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland
^d Department of Surgery, Division of Surgical Oncology, The Johns Hopkins Medical Institutions, Baltimore, Maryland
^e Department of Thoracic Surgery, The Cleveland, Clinic, Cleveland, Ohio

Accepted for publication May 12, 2005.

^_* Address reprint requests to Dr Brock, 240 Blalock, 600 N Wolfe St, Baltimore, MD 21287 (Email: mabrock{at}jhmi.edu).

Presented at the Poster Session of the Forty-first Annual Meeting of The Society of Thoracic Surgeons, Tampa, FL, Jan 24–26, 2005.

	Abstract

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
BACKGROUND: Currently, 40% of patients remain undiagnosed after routine cytologic examination for malignant pleural effusions. Deoxyribonucleic acid (DNA) methylation is a robust strategy for detecting cancer early in tissue. We hypothesized that DNA methylation would be more sensitive in diagnosing patients with malignant pleural effusions than cytology.

METHODS: We conducted a prospective cohort study of 31 inpatients with pleural effusions (24 malignant pleural effusions metastatic from 10 different organs and 7 benign) over 18 months. Aspirated pleural fluid underwent cytologic examination and DNA extraction for nested methylation-specific polymerase chain reaction (PCR). We assayed for promoter hypermethylation in 8 genes known to be methylated in many cancers. Pleural fluid was considered positive if 2 or more genes were methylated by methylation-specific PCR.

RESULTS: Cytology alone confirmed malignant pleural effusions in 15 of 24 patients (sensitivity 63%), whereas methylation alone positively identified 16 of 24 patients (sensitivity 67%). Both tests had 100% specificity in predicting benign effusions. If cytology and methylation were considered together, they exhibited 88% sensitivity and 100% specificity in discriminating benign and malignant effusions. Combined, the two assays were more sensitive than either test alone. Although the positive predictive value of each test was 100%, the negative predictive value of cytology and methylation combined was 78%, better than 47% and 44% for methylation and cytology alone, respectively.

CONCLUSIONS: Epigenetic analysis of pleural fluid can detect malignant DNA from a variety of neoplasms, provide complementarity with cytology, and improve the diagnostic yield of the current standard examination of pleural fluid.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The annual incidence of malignant pleural effusions reported in the United States is now estimated to exceed 150,000 cases per year [1]. The initial diagnostic yield of a malignant pleural fluid cytology after a single thoracentesis is approximately 60%, and is dependent on the tumor type and tumor burden in the chest [2–6]. With a negative cytology result in a patient with suspected malignant pleural effusion, standard clinical practice involves a repeat thoracentesis, a pleural biopsy, or thoracoscopy to prove the presence of tumor cells in the pleural fluid. Because the average life expectancy of a patient with a malignant pleural effusion is only about 3 to 6 months [4, 7, 8], it is imperative for expediency in diagnosis and treatment to ensure a high quality of life. Thoracoscopy is effective in this regard and has a high diagnostic yield for malignant disease involving the pleura [9–12]. Nevertheless, the risks of additional diagnostic procedures that are painful and invasive, such as thoracoscopy, present a considerable clinical challenge. Recent less invasive innovations, such as metabolic imaging with 18-fluro-deoxy glucose positron emission tomography (FDG-PET), are being used as an adjunct in diagnosis, and PET is superior to computed tomography scanning in the detection of pleural metastases with a sensitivity approaching 90%. But a positive reading cannot reliably differentiate at present neoplastic from inflammatory pleural disease [2].

A generally accepted view is that despite 40% of malignant effusions having a negative cytology, there exists in the pleural fluid cancer cells that escape light microscopic detection [4]. Several investigators have attempted to enhance the diagnostic performance of histomorphologic analysis by assaying protein markers [13, 14], but only with limited success. Tumor-specific epigenetic silencing due to hypermethylation in the promoter regions of many genes has been widely documented [15–17]. Because this aberrant DNA methylation can be an early event in tumor progression, it has the potential of being exploited clinically as a marker of early detection [18–20]. There are no current reports evaluating the role of epigenetic markers as a clinical tool to improve the diagnostic yield of patients who present with a de novo pleural effusion. We performed multiplex, nested DNA hypermethylation analyses on prospectively collected pleural fluids in order to investigate if there was a complimentarity of cytology and methylation assays in detecting malignant tumor cells in the fluids. We hypothesized that epigenetic markers would be able to detect patients with malignant DNA in their pleural fluid and that, used with cytology, they would enhance diagnosis.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Clinical and Pathologic Features of the Study Population
Approval to perform this study was obtained from the Institutional Review Board of The Johns Hopkins Medical Institutions. The study population comprised 31 patients with documented pleural effusions who underwent pleural fluid aspiration for diagnostic purposes at The Johns Hopkins Hospital between December 2002 and June 2004. Results of conventional cytology and methylation analyses of the pleural fluids from patients were compared with respect to the definitive diagnosis established by either tissue biopsy, or through clinical follow-up in the case of patients with benign disease. Patients who were free of malignancy had a median follow-up of 6.3 months (range, 0.17 to 25.4). The median age of the study population was 61 years (range, 32 to 83) and there was a slight predominance of males (21 of 31, or 68%). African-American patients comprised 36% (11 of 31) of the cohort. Pleural invasion was defined as invasion of tumor through either the parietal or visceral pleura.

Pleural fluid was obtained either via a needle during thoracentesis (42%), a chest tube during thoracostomy (45%) or by aspiration through a 1-cm incision at the very beginning of a thorascopic pleurodesis procedure (13%). In the 4 patients who underwent thorascopy, the pleural fluid was obtained when there was only one small incision in the chest located in the fifth intercostals space and mid to anterior axillary line. Aspirated pleural fluid was collected in sterile tubes without anticoagulant and rapidly brought to both The Johns Hopkins Hospital pathology laboratory for conventional cytologic examinations as well as to the research laboratory where they were spun, genomic DNA extracted, and DNA hypermethylation analysis performed using multiplex, nested methylation specific polymerase chain reaction (PCR [MSP]) [15, 21]. Cytologic and molecular analyses were performed independently of each other and those carrying out the MSP tests were blinded to the definitive diagnosis of the patients. Cell counts and pleural fluid chemistries were available on 23 patients (74%).

Conventional Cytology
About 100 cc of aspirated pleural fluid was spun in a centrifuge at 2,160 rpm for 10 minutes, supernatant removed, and the cell pellet both preserved onto glass slides in ethanol for Papnicolau staining as well as in formalin and ethanol for processing into cellblocks as per standard protocol in the hospital pathology laboratory. The presence or absence of malignant cells in the cytologic material was reported with the agreement of an attending cytopathologist.

DNA Preparation
An aliquot of about 100 cc of pleural fluid was spun down in a centrifuge at 2,000 rpm for 10 minutes, and the supernatant collected. Genomic DNA from the supernatant was extracted by standard methods using a simplified proteinase K digestion method. The DNA was dissolved in low TE buffer and stored at -20°C.

Methylation-Specific PCR
Genomic DNA from pleural fluid was bisulfite modified as previously described [21]. The MSP primers were designed according to genomic sequences flanking the presumed transcription start sites for all genes and are located as described [22]. Primer sequences were oligosynthesized (IDT) to allow MSP to detect bisulfite-induced changes affecting unmethylated (U) and methylated (M) alleles. A multiplex-nested MSP assay as previously described was used for all samples [23]. The nested approach amplifies bisulfite-modified DNA initially with flanking PCR primers without preferentially amplifying methylated or unmethylated DNA. The resulting fragment is then used as the template for MSP. Primer sequences of p16, MGMT,BRCA1,APC, RASSF1A, RARß, FHIT, and CRBP1 have all been previously described [24–28]. Each MSP reaction incorporated approximately 100 ng of bisulfite-treated DNA as template, 10 pmoles of each primer, 100 pmoles dNTP, 10X PCR buffer, and 1 unit of JumpStart Red Taq Polymerase (Sigma-Aldrich, St. Louis, Missouri) in a final reaction volume of 25 µL. Placental DNA treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, Massachusetts) was used as a positive control for the remaining genes. The DNA from normal lymphocytes was used as a negative control for methylated genes. The annealing temperature for all reactions was 60°C. The PCR products were analyzed as previously outlined [17]. Cycle conditions were as follows: 95°C x 5 minutes; 35 cycles x (95°C x 30 seconds, 60°C x 30 seconds, 72°C x 30 seconds); 72°C x 5 minutes. The MSP products were analyzed using nondenaturing 6% polyacrylamide gel electrophoresis, stained with ethidium bromide and visualized under ultraviolet illumination. Efficiencies of the methylation reactions were controlled in each analysis by including methylated and unmethylated control DNA. All assays were performed in triplicate to confirm results.

Bisulfite Genomic Sequencing
Because DNA methylation occurs primarily during carcinogenesis, bisulfite genomic sequencing of the methylated-PCR product samples from patients with benign disease was performed to ensure that no technical errors due to false priming had occurred. Bisulfite-treated DNA was subjected to PCR using primers flanking the targeted MSP regions for p16, APC, RARß, and MGMT. The sequencing primers were as follows: p16-forward, 5'-TTATTAGAGGGTGGGGTGGATTGT-3'; p16-reverse, 5'-CAACCCCAAACCACAACCATAA-3', APC-forward, 5'-GTGTTTTATTGTGGAGTGTGGGTT-3'; APC-reverse, 5'-CCAATCAACAAACTCCCAACAA-3'; RARß-forward, 5'-TTGGGATGTTGAGAATGTGAGTGATTT-3'; RARß-reverse, 5'-CTTACTCAACCAATCCAACCAAAACAA. The conditions for PCR were as follows: 95°C x 5 minutes; 35 cycles x (95°C x 30 seconds, 56°C x 50 seconds, 72°C x 50 seconds); 72°C x 5 minutes. The PCR products were gel purified, and then sequenced with the M13 reverse primer by automated sequencing (The Johns Hopkins University School of Medicine Biosynthesis and Sequencing Facility, Department of Biological Chemistry, Baltimore, Maryland).

Statistical Analysis
Sensitivity, specificity, positive predictive values and negative predictive values of both MSP and conventional cytology as well as of the combination of both techniques were calculated in relation to the definitive diagnosis of the patients in the study. Summary receiver operating characteristic curves, which can analyze the accuracy of a single test in a single population, could not be employed because the specificity of both the cytologic and methylation tests was 100%. Correlation between variables was estimated using the Fisher's exact test, or the ² test when appropriate. All p values are two-sided, and all significant associations were considered when the p value was 0.05 or less (Stata Statistical Software, College Station, Texas).

	Results

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Of the 24 patients with malignancy as the definitive diagnosis, 10 distinct primary tumors from 10 different tissues of origin were included in the study (Table 1). Primary cancers from the lung and breast represented the overwhelming majority of malignancies: 16 of 24 (67%).

Correlation of Cytology and Methylation Analyses of Pleural Fluid
Although both cytology and methylation analysis were comparable in terms of sensitivity, the tests were independent of each other. Hypermethylation identified 10 of 15 pleural fluid aspirates (67%) that were positive by cytologic analysis (Fig 1). The primary tumors of origin for the fluids that were positive by cytology but not detected by epigenetic analysis were a peripheral nerve sheath tumor, a melanoma, a mesothelioma, and two nonsmall-cell lung (NSCLC) carcinomas. In addition, DNA methylation detected the presence of methylated DNA in the fluid from 6 of 9 patients (67%) who had cytology negative aspirates (Figs 2 and 3). In 3 patients (1 patient with acute myelocytic leukemia, another with renal cell carcinoma, and the third with NSCLC), cytology and methylation as a combined analysis failed to detect malignant cells or methylated DNA, respectively. Of the 7 patients with benign disease, 1 patient (status post two previous heart transplants and one renal transplant) was strongly methylated for the RARß gene, and 2 patients (1 with Mediterranean Fever and the other with chronic obstructing pulmonary disease) had weakly methylated promoter regions for MGMT and APC, respectively. Bisulfite genomic sequencing confirmed that all three of the genes found methylated by MSP in the patients without cancer had persistence of "Cs" preceding "Gs" after bisulfite modification indicating methylated cytosines. These sequencing data indicate that the positive methylation result in the patients with benign disease was not due to technical limitations.

View larger version (17K): [in this window] [in a new window]   Fig 1. Comparison between DNA methylation and cytology positive pleural fluid. Pleural fluid was methylation positive if two or more genes were strongly methylated. In a semiquantitive methylation score, percent methylation was calculated as follows: strongly methylated 1/8 (black), weakly methylated 0.5/8 (gray), and no methylation 0/8 (white). The test was considered negative if methylation was 18.8% or less. (1 = positive test; 0 = negative test; PF = pleural fluid.)

View larger version (18K): [in this window] [in a new window]   Fig 2. Comparison between DNA methylation and cytology negative pleural fluid in patients with metastatic primary tumors. Pleural fluid was methylation positive if two or more genes were strongly methylated. Percent methylation was calculated as follows: strongly methylated 1/8 (black), weakly methylated 0.5/8 (gray), and no methylation 0/8 (white). The test was considered negative if 18.8% or less methylated. (1 = positive test; 0 = negative test; PF = pleural fluid.)

View larger version (18K): [in this window] [in a new window]   Fig 3. Comparison between DNA methylation and cytology negative benign pleural fluid was ascertained as methylation positive if two or more genes were strongly methylated. Percent methylation was calculated as follows: strongly methylated 1/8 (black), weakly methylated 0.5/8 (gray), and no methylation 0/8 (white). The test was considered negative if 18.8% or less methylated. (1 = positive test; 0 = negative test; IVD = in vitro methylated control; NL = normal lymphocytes; PF = pleural fluid.)

Correlation of Diagnostic Tests with Pleural Invasion
Eighteen of the 24 patients with malignancy (75%) had documented invasion of their pleura by histopathology (Table 2). Neither a positive cytologic nor a positive methylation test alone correlated with the presence of malignancy in the pleura. When the two techniques are combined, however, a positive result predominates in those patients with invasion of the pleura (p = 0.05).

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Molecular analysis of the pleural fluid of patients using DNA hypermethylation to discriminate those with benign and malignant pleural effusions has not been well studied. We hypothesized that in patients in whom malignant cells may not be detectable in the pleural fluid by conventional cytology, free-floating, aberrantly hypermethylated DNA would be present in sufficient quantities to be detected, and that the sensitivity of this approach would improve the overall diagnostic sensitivity of conventional cytology. Our data show that even for multiple tumor types, and with a limited cohort size, we were able to improve the diagnostic performance of cytologic analysis by measuring semiquantitatively gene hypermethylation markers in the pleural fluid. We employed a panel of eight genes and a multiplex, nested MSP technique and observed that conventional cytology and methylation analysis alone have similar sensitivities (63% and 67%, respectively), but that the sensitivity improves to 88% when the results of the methylation and cytology techniques are combined.

We have observed previously that a single methylation marker is invariably insufficient to characterize fully malignancies from many organ sites, and that a panel of epigenetic markers is essential to provide a spectrum wide enough to detect all types of malignancies [26]. In addition, we have realized that certain genes are preferentially methylated depending on the tumor's organ of origin producing a unique epigenetic fingerprint [26]. Using a sole methylation marker, p16, to evaluate intraoperatively postresectional pleural lavages for tumor DNA, Ng and coworkers [29] had a sensitivity of 29% (4 of 14) in detecting malignant DNA. Although we had tumors from 10 different organ sites, we designed a broad panel of genes to detect primarily breast and lung tumors since the most common malignancies that metastasize to the pleural fluid are, in order of frequency, lung, breast, and lymphoma [4, 6]. Our gene panel was successful in detecting breast and lung neoplasms with sensitivities of 100% and 75%, respectively. Moreover, 5 of the 8 tumors that methylation alone failed to detect were not covered by our panel, namely, a peripheral nerve sheath tumor, melanoma, renal cell, acute myelocytic leukemia, and mesothelioma. Indeed, it will be a challenge to be borne out of subsequent studies to determine how large of a panel of methylated genes will be required to identify the variety of neoplasms that metastasize to the pleural cavity.

We also observed in the present study that pleural fluids positive by the combined methylation and cytology analysis correlated with pleural invasion. These data suggest that there is a higher yield of both tumor DNA and sloughed malignant cells in the pleural fluid after the pleura is invaded by tumor. This is in agreement with recent studies performed using pleural lavages during pulmonary resections which have shown increased detection of positive pleural lavage cytology when there is parietal pleura invasion by malignancy [30]. Combined cytology and methylation analysis were also more accurate in discerning exudative samples from patients with a primary malignancy. In general, exudative malignant pleural effusions have higher cell counts, lower glucose and pH levels, and are cytologically positive. But these criteria are far from absolute [4], and in our dataset, cytologic analysis only detected 57% of exudative pleural fluids from patients with a known primary malignancy compared with 64% for MSP alone and 79% for the methylation and cytology assays combined. It is interesting to speculate that the addition of methylation analysis may increase the sensitivity of conventional cytology because tumor DNA may more easily enter the pleural fluid than shed malignant cells, such as by transudation from the serum. Numerous studies have demonstrated the presence of circulating tumor-specific hypermethylated DNA in the serum of patients with a variety of tumors, such as esophagus, gastric, head and neck, NSCLC, hepatic, and prostate malignancies [31–35]. The serum levels may range from 10 to 30 ng/mL of normal DNA in control subjects to 30 to 1,200 ng/mL in cancer patients [36–38]. In the main, tumor DNA levels in these studies are several-fold higher in the serum of patients with malignancy than in matched plasma samples of healthy controls [36].

Limitations of the present study include the small cohort size that precludes detailed analysis of specific tumor subtypes. Only descriptive, observational conclusions about the methylation analyses in patients with NSCLC and breast cancer can be made. Second, there was an absence of corresponding epigenetic profiling being performed on the primary tumor from each patient in the study. These results may have added internal validity if the primary tumors had similar methylation profiles as the malignant pleural fluid. A parallel epigenetic study of the primary tumors would also have been useful in interpreting the pleural fluids of patients with only one gene methylated by MSP. Rather than a technical limitation, the lack of methylated DNA in the pleural fluid in these patients may reflect the absence of methylated markers in the primary tumors. Third, we arbitrarily defined 2 of 8 genes being strongly methylated as the criterion for a positive methylation test. Aberrant promoter methylation and transcriptional repression is often an early event in carcinogenesis, and we as well as others have observed methylation in histologically normal tissue [15, 39, 40]. Although we used in this study a semiquantitative approach to discriminate a true-positive result from preneoplastic methylation events, based on a percentage of methylated genes in a panel, a more robust quantitative PCR approach [41] for detecting methylated tumor DNA in the pleural fluid may also be useful. Finally, the patients with benign disease in this study only had 6 months of longitudinal follow-up to determine if they remained cancer free. That is usually insufficient time to allow preneoplastic events to progress to clinically detectable cancer.

Despite our ability to detect methylated DNA in pleural fluid, the sensitivity of this assay would most likely be enhanced by designing separate gene panels for tumors originating from specific organs. Epigenetic profiles of tumors from different organs do seem to be distinct [26], but more validity studies are necessary before these profiles can be optimized for routine clinical diagnostic use. Improving the diagnostic performance of any test has the potential to alter medical practice, even if, as in this case, it simply means eliminating the need for repeated thoracentesis or other invasive procedures to secure the diagnosis of metastatic disease in a patient with a know primary. Although a more sensitive test to diagnose patients earlier may have limited clinical value in extending survival in patients with metastatic disease, this assay may have important diagnostic implications particularly in patients who have unilateral pleural effusions as their principal presenting clinical sign. It is conceivable that methylation profiles of the pleural fluid could also be used to identify the site of origin of the metastatic DNA in the chest. But this technology requires much validation before it becomes clinically widespread. At present, commercial availability of MSP kits is limited. We estimate, however, that in a high throughput clinical laboratory, the costs to perform MSP on the pleural fluids in this study would be about $20 to $30 per clinical sample for reagents.

In conclusion, this study shows the feasibility of detecting neoplastic methylated DNA from a variety of different malignancies in the pleural fluid of patients even if conventional cytologic analyses are negative. In addition, the methylation test may increase the sensitivity of conventional cytology without a corresponding decrease in specificity. Larger translational studies will be needed to validate that epigenetic markers may improve the diagnostic yield of the current standard examination of pleural fluid.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This work has been generously supported in part by the National Cancer Institute, Early Detection Research Network Grant CA84986, and by the National Cancer Institute Specialized Program of Research Excellence (NCI/SPORE) Grant CA58184.

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Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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