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A Comparison of Cytology and Fluorescence in Situ Hybridization for the Detection of Lung Cancer in Bronchoscopic Specimens^*

^* From the Mayo Clinic, Departments of Laboratory Medicine and Pathology (Drs. Halling, Kipp, and Harwood) and Medicine (Drs. Rickman and Jett), Rochester, MN; and Department of Medicine (Dr. Doerr), Division of Pulmonary and Critical Care, Baylor College of Medicine, Baylor, TX.

Correspondence to: Kevin C. Halling, MD, PhD, Hilton 920B, Mayo Clinic, Rochester, MN 55905; e-mail: halling.kevin{at}mayo.edu

Abstract

Study objectives: To determine the relative sensitivity and specificity of cytology and fluorescence in situ hybridization (FISH) for the detection of lung cancer in bronchoscopically obtained specimens.

Design: Cytology and FISH were performed on brushing and washing specimens obtained from patients undergoing bronchoscopy for suspected lung cancer. FISH utilized the LAVysion probe set (Abbott Molecular; Des Plaines, IL), which contains locus-specific probes to 5p15, 7p12 (EGFR), 8q24 (C-MYC), and a centromeric probe to chromosome 6.

Setting: Single-center, academic, tertiary medical center.

Participants: One hundred thirty-seven patients referred for bronchoscopy for suspicion of lung cancer.

Interventions: Cytology and FISH were performed on bronchoscopic brushings and washings.

Measurements and results: One hundred thirty-seven patients undergoing bronchoscopy had pathology, FISH, and cytology results. FISH and cytology were performed on 123 washing and 78 brushing specimens. Sensitivities of FISH and cytology were 71% and 51% (p = 0.007), respectively, for brushing specimens, and 49% and 44% (p = 0.541) for washing specimens. When FISH and cytology results were combined, sensitivities were 75% and 61%, respectively, for brushing and washing specimens, which was significantly better (p < 0.001) than cytology alone. Specificities of FISH and cytology for patients with negative findings at the time of initial bronchoscopy were 83% and 100% (p = 0.125), respectively, for brushing specimens, and 95% and 100% (p = 0.500) for washing specimens.

Conclusions: These findings show that FISH is significantly more sensitive than conventional cytology for detecting lung cancer in bronchial brushing specimens; when combined with cytology, FISH can improve the diagnostic sensitivity of detecting malignancy in bronchial brushing and washing specimens.

Key Words: bronchial brush • bronchial washings • bronchoscopy • fluorescence in situ hybridization • lung neoplasm

In 2005, approximately 172,500 people in the United States will receive a diagnosis of lung cancer.¹ The dismal 5-year survival of 15% for all patients with lung cancer has been attributed to the fact that most patients have advanced disease at the time of diagnosis.¹²³ Cytology (brushings and washings) and biopsy specimens collected during bronchoscopy are widely used techniques to diagnose malignancy in patients suspected of having lung cancer.⁴ Previous studies⁵ have shown that in centrally located tumors, the diagnostic sensitivities of bronchial brushing and washing specimens range from 44 to 94% (mean, 72%) and 27 to 90% (mean, 68%), respectively. However, the diagnostic sensitivity of both cytologic techniques drops considerably in peripheral tumors, where sensitivities range from 6 to 83% (mean, 45%) in brushings, and 4 to 43% (mean, 28%) in washings.⁵ These data illustrate the need for improved clinical and/or laboratory tests to accurately detect disease, especially in peripherally located tumors.

Fluorescence in situ hybridization (FISH) is a technique that utilizes fluorescently labeled DNA probes to detect chromosomal abnormalities in cells. FISH has been used clinically to detect bladder cancer cells in urine,⁶⁷⁸ and biliary tract malignancy in endoscopic retrograde cholangiopancreatography-obtained biliary brushing specimens.⁹ The LAVysion multicolor, DNA-based FISH probe (Abbott Molecular; Des Plaines, IL) is a probe developed originally for the detection of lung cancer.¹⁰ The LAVysion probe contains locus-specific probes to 5p15, 7p12 (EGFR), 8q24 (C-MYC), and a centromeric probe to chromosome 6. Studies show that FISH, using this probe set, is useful for the detection of lung cancer in bronchial washings,¹¹ bronchial brushings,¹¹¹² and sputum.¹³ However, no large prospective studies have been performed to evaluate the utility of the LAVysion probe for detecting centrally and peripherally located early and late stage cancers in bronchial brushing and washing specimens. The goal of this study was to prospectively determine the relative sensitivity and specificity of conventional cytology and FISH for the detection of lung cancer in specimens obtained from patients undergoing bronchoscopy for suspected lung cancer.

Methods and Materials

Patients and Specimens
As approved by the Mayo Clinic Institutional Review Board, 137 patients undergoing bronchoscopy for suspected lung cancer consented to and were enrolled in the study. When possible, brushing and washing specimens were collected, and both cytology and FISH analysis were performed. Brush specimens were obtained using standard bronchoscopic techniques. Four smears were prepared from the brushing specimens. One slide was used for FISH, and the remaining three slides were used for routine clinical cytology. When possible, washings were obtained by introducing sterile saline into the airway and collecting the fluid. This fluid was split evenly for cytology and FISH analysis.

Sample Preparation for FISH
Bronchial brushing specimens were received in the cytology and FISH laboratories as ethanol spray-fixed smears. No additional slide preparation was required prior to FISH hybridization for these specimens. Washing specimens were collected in 50-mL conical tubes and were frequently bloody. The 50-mL tube was centrifuged at 400g for 10 min and the cell pellet transferred to a 15-mL conical tube for further processing. To lyse the RBC, ammonium chloride (ACK) lysing solution (150 mmol/L NH₄Cl, 1 mmol/L KHCO₃, 0.1 mmol/L Na₂ ethylenediamine tetra-acetic acid [pH 7.3]) was added to the tube (filled to 13mL, final volume of specimen and ACK solution) and allowed to sit at room temperature for 10 min. One milliliter of 3:1 methanol:acetic acid fixative was then added to the conical tube containing specimen and ACK lyse solution, vortexed, and centrifuged at 400g for 8 min. After removing the supernate, approximately 10 mL of 3:1 methanol:acetic acid fixative was added. This cell pellet wash was repeated three times. After the final centrifugation, the supernatant was aspirated, leaving 1.5 to 2.0 mL of 3:1 fixative in the conical tube. The cell pellet was then resuspended and transferred to a 1.8-mL microcentrifuge tube. Ten microliters of the cell suspension was then placed on a 1-cm, etched-ring slide (Gold Seal, No. 12–568-20; Fisher Scientific; Pittsburgh, PA) and allowed to dry. The density of cells on the slide (ie, cellularity) was then assessed with a phase-contrast microscope. Additional cell suspension was applied to the slide if the cellularity was low. The optimal cellularity was considered to be the maximum number of cells without significant cell overlap on the slide.

FISH Prehybridization and Hybridization
Brushing slides were immersed in fresh 3:1 methanol:acetic acid fixative for 10 min. This helped to remove excess debris and prepare the specimen for hybridization. This step was not required for washing specimens. The pretreatment and hybridization procedures were otherwise identical for both specimen types and were essentially performed as previously described.¹⁴ Minor deviations to the previously described procedure included the substitution of 2 x saline sodium citrate (SSC) at 37°C for 10 min for 2 x SSC at 80°C for 2 min as part of the prehybridization treatment and the use of a 0.005% pepsin solution rather than a 0.05% pepsin solution, 2,500 to 3,500 U/mg (Sigma; St. Louis, MO).

The LAVysion probe set contains locus-specific probes to 5p15, 7p12 (EGFR), 8q24 (C-MYC), and a centromeric probe to chromosome 6. Ten microliters of LAVysion probe (Abbott Molecular) was applied to a 22 x 22 mm area of the slide for the brushing specimens, and 3 µL of LAVysion probe was used for the washing slide specimens. Specimen DNA and probe DNA were co-denatured and hybridized using a Vysis HYBrite instrument (Abbott Molecular) set at a denaturation temperature of 73°C for 3 min and a hybridization temperature of 37°C for 10 to 16 h. Following hybridization, the slides were washed in 2 x SSC/0.1% NP-40 at 76°C for 1 min. Counterstain (4'-6-diamidino-2-phenylindole [DAPI]; Abbott Molecular) was then applied, and the slides were coverslipped for analysis.

FISH Scoring
Cases were analyzed without prior knowledge of the biopsy, cytology, or clinical findings. Slides were assessed by scanning for cells with cytologic atypia (larger nuclei, irregular shape, mottled, or patchy DAPI staining) using a single-bandpass DAPI filter, and the FISH signals were then assessed and recorded in these cells.¹⁵ This scanning algorithm is identical to the scanning algorithm that is utilized to scan slides for urothelial carcinoma cells with the UroVysion assay.⁶¹⁴¹⁶ However, we also analyzed a fraction of the nuclei without cytologic atypia in each field of view when scoring LAVysion to ensure that we were not missing normal-appearing cells with chromosomal abnormalities.

Criteria for FISH Abnormality
Two types of chromosomal abnormality (polysomy and tetrasomy) were assessed with the LAVysion probe set. Gain was defined as three or more signals for a given probe. A polysomic cell was defined as a cell with gains of two or more of the four probes (Fig 1 , top, A). Tetrasomic cells demonstrate four copies for each of the four probes (Fig 1, bottom, B). Based on previous data, a case was considered positive for malignancy if five or more of the cells, regardless of the total number of cells on the slide, exhibited polysomy.¹⁰ For tetrasomy, 10 cells were needed for the case to be considered positive.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Representative examples of cases demonstrating polysomy (top, A) and tetrasomy (bottom, B). Polysomic cells show gains (ie, three or more signals) for two or more of the four probes but do not include tetrasomic cells. Tetrasomic cells have four copies of each of the four probes.

Statistics
The "gold standard" used for calculation of sensitivity and specificity was bronchoscopic biopsy, surgical resection, or transthoracic needle aspirate tissue diagnosis at the time of or within 2 weeks of the bronchoscopy that provided the specimens for cytology and FISH analysis. Cytology cases interpreted as equivocal but not diagnostic for cancer were treated as negative for malignancy for sensitivity and specificity calculations. The McNemar test for correlated proportions was used to determine the difference in sensitivity and specificity between FISH and cytology.⁸ Early and late stage tumors were defined as stages I-IIIA and stage IIIB-IV, respectively.

Results

Ninety-two men and 45 women consented to this study (mean age, 66 years; range, 17 to 86; median, 69). Of the patients enrolled in this study, 119 patients (87%) underwent bronchoscopic biopsy, 48 patients (35%) had transthoracic needle aspirates, and 31 patients (23%) were sent for surgical resection following bronchoscopy. A summary of the pathologic findings for the 89 patients with tumor are shown in Table 1 . The remaining 48 patients did not have pathologic evidence of malignancy. Since the histopathologic findings alone often did not provide enough evidence describing what the overall diagnosis was, we utilized not only the histopathologic but also the radiologic and other clinical findings to categorize the diagnoses for the 48 patients without evidence of tumor. Of these 48 patients, 13 were being evaluated for evidence of recurrent tumor or metastatic disease from another site and were not found to have tumor. The diagnoses for the remaining 35 patients were as follows: pneumonia (n = 10), sarcoidosis (n = 5), infection (n = 4), pulmonary embolism (n = 2), lung abscess (n = 1), chronic granulomatous disease (n = 1), histiocytosis X (n = 1), bronchial stenosis with atelectasis (n = 1), and indeterminate (n = 10).

There were no significant differences in the specificity of cytology and FISH results in bronchial brushing and washing specimens (Table 3). The specificities of the bronchial washing specimens for cytology and FISH were 100% and 95% (p = 0.500), respectively.

Discussion

The results of this study demonstrate that FISH is more sensitive than conventional cytology for detecting lung cancer in bronchial brushing specimens; when combined with cytology, FISH can improve the diagnostic sensitivity of detecting malignancy in bronchial brushing and washing specimens. The brushing data show that FISH was able to increase the diagnostic sensitivity of detecting peripheral tumors over routine cytology. The data also suggests that FISH and cytology brushing results combined can improve the detection of early stage lung cancers (stages I, II, and IIIA) over cytology alone.

The brushing sensitivities of 63% for FISH and 33% for cytology on peripheral lesions observed in this study (Table 4) are similar to the sensitivities of 68% and 39% for FISH and cytology on peripheral brushing specimens reported by Bubendorf et al.¹¹ The increased diagnostic sensitivity of FISH brushings relative to cytology is important since flexible bronchoscopy has poor sensitivity for peripheral tumors.¹⁷ An explanation that was offered by Bubendorf et al¹¹ for the higher sensitivity of FISH for peripheral tumors is that pathologists may be unable to make an unequivocal diagnosis of malignancy in cytologic specimens in which cells with reactive or atypical features are covered in mucinous and/or cellular debris. Our data provide some support for this possibility since 5 of the 13 cancer patient brushings with FISH positive and false-negative cytology results were actually diagnosed as equivocal by cytology. Another possible explanation for the higher sensitivity of FISH for peripheral tumors may be that FISH has an ability to detect small populations of tumor cells that are distant from the primary tumor. Patients who acquire lung cancer frequently have "field cancerization" defects in which there are extensive areas of visually normal-appearing mucosa that have genetic abnormalities.¹⁸ It is postulated that one or more of the cells in this area of field defect eventually go on to form the predominant tumor. Perhaps FISH is detecting cells that are part of the early field defect but not necessarily cells from the bronchoscopically targeted tumor.

The findings of this study suggest that FISH and cytology brushing results combined can improve the detection of early stage lung cancers (stages I, II, and IIIA) over cytology alone. This is clinically important since these patients often benefit from surgery and a negative or nondiagnostic bronchoscopy may delay an attempt at curative surgery.¹⁹²⁰²¹ Of the 25 FISH brushing specimens from patients who were found to have early stage disease, FISH was able to detect four more lung cancers (16%) than cytology; and when the FISH and cytology results were combined, malignancy was diagnosed in an six additional patients than with cytology alone (24%). These findings suggest that FISH and cytology complement one another in the detection of lung cancer in brushing specimens. The finding that FISH and cytology complement one another in brushing specimens has also been previously reported.¹¹

Increased sensitivities of tests are often associated with decreases in specificity, which can ultimately reduce the clinical utility of these tests.²² In the current study, the specificity of FISH appeared to be lower than cytology for brushes (100% vs 83%, p = 0.125), although this difference did not reach statistical significance. We reviewed the clinical histories of the four patients with apparent false-positive FISH brushing results (ie, patients with a negative evaluation for tumor at the time of bronchoscopy but a positive FISH result) to investigate the nature of these apparent false-positive results. This revealed that one patient had a surgical resection for stromal and bronchoalveolar lung cancers prior to the bronchoscopy and was receiving therapy at the time of bronchoscopy. The remaining three patients had no previous history of lung cancer and no current evidence of lung cancer (follow-up time, 6 months, 2 years, and 2 years, respectively). All four of the cytology results for these specimens were also negative for malignancy. In the washing specimens, there were two FISH false positives. Follow-up on these two patients revealed that one of the patients had undergone surgical resection for lung cancer prior to the bronchoscopy and was receiving therapy, and the other patient has metastatic colon cancer to the liver and progressive pulmonary infiltrates. Both of these specimens were also negative by cytology.

Possible explanations for false-positive FISH results could be that FISH is able to detect malignancy before there is clinical or pathologic evidence of cancer,²³ FISH is able to detect chromosomal abnormalities in high-risk patients without cancer,²⁴²⁵ a nonmalignant clinical entity (eg, an infectious process) is causing chromosomal alterations that are detectable with the probe set,²⁶ or the threshold of five polysomic cells used to consider a case positive is too low.²⁵ Other studies¹⁰¹¹ using the LAVysion probe have found similar specificities (87% and 82%) with five or six cell cutoffs. Larger studies with extended follow-up will be required to determine if apparent false-positive LAVysion FISH results represent true false-positive results or an ability of the FISH probe set to detect tumor before it is detectable by other modalities. A study²⁷ with a bladder cancer detection FISH probe set have demonstrated that it is frequently able to detect tumor months before it is detectable by cystoscopy.

The question then becomes how to manage patients with a positive FISH result but a negative cytology result. It is difficult to make firm recommendations because of the small number of patients studied to date. However, until larger studies are performed we feel that in the absence of corroborating cytology and/or biopsy findings a positive FISH result should raise the index of suspicion for cancer but not lead to surgical or chemotherapeutic treatment. Individual patients should be followed up closely and reassessed for signs or symptoms of cancer at regular intervals (eg, every 6 months). Fluorescence bronchoscopy might be a technique that could detect occult disease in such patients.²⁸

In the current study, there was not a significant difference (p = 0.541) in the sensitivities of FISH and cytology for detecting malignancy in bronchial washing specimens (Table 3). However, by combining the FISH and cytology results, 14 additional patients (17%) were identified as having malignancy. This increase in sensitivity was accomplished while maintaining high specificity (95%).

Disadvantages of FISH relative to cytology include that it is more expensive than cytology and that it cannot assist clinicians in determining the histologic subtype of the tumor. However, the greater ability of FISH to detect early and peripheral disease than cytology could lead to improved overall survival. Since cytology is less expensive than FISH and has high specificity, the most cost-efficient way to utilize FISH in clinical practice may be to reflex cytology equivocal and/or negative cases to FISH. Since most laboratories utilize liquid-based cytology, it should be possible for most laboratories to prepare additional slides for ancillary studies such as FISH when necessary.

Although the LAVysion probe was primarily designed to assist in the detection of lung cancer in cytologic specimens, it also has the potential to provide additional prognostic and therapeutic information that cannot be obtained with conventional cytology. For example, one of the four probes in the LAVysion probe set, the LSI 7p12 probe, hybridizes to the epidermal growth factor receptor locus. A recent study²⁹ has shown that increased epidermal growth factor receptor copy number detected by FISH is associated with improved survival after treatment with gefitinib. Alterations of the 8q24 (C-MYC) and 20q loci that are represented in the LAVysion probe set might also have prognostic or therapeutic significance.³⁰³¹ While a number of studies^10111213 now suggest that FISH improves the sensitivity of lung cancer detection over cytology, further studies are needed to determine the proper algorithm for using FISH in the clinical laboratory.

Conclusion

This study demonstrates that FISH is significantly more sensitive than conventional cytology for detecting lung cancer in bronchoscopically obtained bronchial brushing specimens. FISH detected tumors at earlier stages and detected a higher proportion of peripheral tumors. Additional studies are needed to determine if the higher sensitivity of FISH has the potential to improve patient survival.

Acknowledgements

Becky Kruger, RN, for specimen collection and distribution; personnel from the Molecular Cytology and Imaging Laboratory; and members of the bronchoscopy group for specimen collection: Drs. Eric S. Edell, David E. Midthun, Udaya B.S. Prakash, Karen L. Swanson, and James P. Utz.

Footnotes

Abbreviations: ACK = ammonium chloride potassium; DAPI = 4'-6-diamidino-2-phenylindole; FISH = fluorescence in situ hybridization; SSC = sodium chloride-sodium citrate

This study was supported in part by a grant from the ACCP/Lungevity Foundation and Vysis, Inc. Dr. Kevin Halling is listed as a co-inventor on a patent that has been filed for the FISH probe set used in this study. He and Aaron Harwood receive royalties from the sale of this product. In addition, Dr. Halling receives grant support from Abbott Molecular Inc. to develop FISH assays for the detection of malignant cells in cytologic specimens.

Received for publication December 14, 2005. Accepted for publication March 24, 2006.

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This Article Abstract Full Text (PDF) Free Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Article Archive Download to citation manager Citing Articles Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Halling, K. C. Articles by Jett, J. R. Search for Related Content PubMed PubMed Citation Articles by Halling, K. C. Articles by Jett, J. R.