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(Chest. 2005;128:906-911.)
© 2005 American College of Chest Physicians

Quantitative Detection of Lung Cancer Cells by Fluorescence In Situ Hybridization*

Comparison With Conventional Cytology

Haruhiko Nakamura, MD, PhD; Idiris Aute, MD, PhD; Norihito Kawasaki, MD; Masahiko Taguchi, MD, PhD; Tatsuo Ohira, MD, PhD and Harubumi Kato, MD, PhD, FCCP

* From the Department of Chest Surgery (Drs. Nakamura, Kawasaki, and Taguchi), Atami Hospital, International University of Health and Welfare, Atami, Shizuoka, Japan; the Department of Thoracic Surgery (Dr. Aute), The First Affiliated Hospital, Xinjiang Medical University, Urumqi, People’s Republic of China; and the Department of Surgery (Drs. Ohira and Kato), Tokyo Medical University, Tokyo Japan.

Correspondence to: Haruhiko Nakamura, MD, PhD, Department of Chest Surgery, Atami Hospital, International University of Health and Welfare, 13-1 Higashikaigan-cho, Atami-city, Shizuoka, Japan 413-0012; e-mail: h.nakamura{at}iuhw.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: The aim of this study was to clarify whether fluorescence in situ hybridization (FISH) can diagnose lung cancer in various clinical specimens in comparison with conventional cytology.

Design: Prospective study.

Setting: University hospital in a metropolitan area.

Patients: Fifty consecutive patients with abnormal chest radiography or CT scan findings were enrolled. The patients included 32 men and 18 women, with an average age of 64 years. The final definitive diagnosis was made by histologic examination, as follows: 38 primary lung cancers (24 adenocarcinomas, 8 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas); 1 metastatic renal cell carcinoma; and 11 benign lesions.

Methods: Four types of clinical specimens were analyzed. Cells obtained by transbronchial brushing and transbronchial fine-needle aspiration using a fiberoptic bronchoscope under fluoroscopy, CT scan-guided percutaneous needle biopsy, and bronchial washings. On every examination, duplicate slides were made for analyses of conventional cytology and FISH.

Results: Classifications according to conventional cytology were as follows: class I, 4 patients; class II, 15 patients; class IIIa, 3 patients; class IIIb, 5 patients; and class V, 23 patients. A classification higher than class IIIb was considered to be positive for cancer. For cytology, we found no false-positive cases and 11 false-negative cases. The specificity was 100%, and the sensitivity was 71.8%. By FISH, 34 cases showed aberrant copy numbers in either chromosome 3 or 17. We found no false-positive cases and five false-negative cases. The specificity was 100%, and the sensitivity was 87.1%.

Conclusion: The ability of FISH to detect aneusomic lung cancer cells is superior to conventional cytology for the diagnosis of lung cancer.

Key Words: aneuploidy • aneusomy • cytology • fluorescence in situ hybridization • lung cancer


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Conventional cytology plays an important role for the diagnosis of lung cancer, especially in the examination of sputum and pleural effusions. In addition, cell specimens obtained by transbronchial brushing (TB)1 and needle aspiration under fluoroscopy,2 percutaneous needle biopsy (PN) under CT scanning,3 and bronchial washings (BWs)4 provide important information for the differential diagnosis between benign and malignant disease. Cytologic diagnoses are made by experienced cytologists who can properly evaluate the morphologic features of malignant cells. However, this judgment is sometimes difficult when the morphologic changes associated with malignancy are mild. Such cells are usually classified as class III, using the classification of Papanicolaou,5 which is suggestive of, but not conclusive for, malignancy. This is an ambiguous judgment for clinical decision making. In addition, when one obtains a small number of cells from the lesion, the definitive diagnosis is even more difficult. These limitations of morphology-based conventional cytology have stimulated the search for more objective and quantitative methods for an accurate cytologic diagnosis of cancer.

Aneuploidy is the most common feature of many solid tumors, including lung cancer.6 Solid tumors are characterized by complicated karyotypes by classic cytogenetics.78 Chromosomal instability910 may cause the uneven distribution of chromosomes during cell division.1112 Thus, malignant tumors can be diagnosed by detecting aneuploid, usually hyperdiploid, cells. A rapid and sensitive method for detecting aneusomy of a specific chromosome in an individual cell is fluorescence in situ hybridization (FISH). For this purpose, specific centromeric DNA probes enumerated the chromosomes. FISH was originally developed as a method to detect chromosomal aberrations,13 and is now widely used for gene mapping,14 the diagnosis of congenital diseases,15 and detecting specific gene copy number changes in malignant cells.161718

One advantage of FISH in detecting malignant cells is its objective and quantitative evaluation. However, the specificity and sensitivity of FISH in the diagnosis of lung cancer is unclear. We report the results of a prospective study comparing FISH with conventional cytology to detect lung cancer cells.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Fifty consecutive patients who underwent cytologic examination for abnormal chest radiography or CT scan findings at Tokyo Medical University Hospital from July 2003 to January 2004 were enrolled in this prospective study. The patients included 32 men and 18 women, with an average age of 64 years. The final definitive diagnosis was made by histologic examination, as follows: 38 primary lung cancers (24 adenocarcinomas, 8 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas); 1 metastatic renal cell carcinoma; and 11 benign lesions. All patients with lung cancer were staged according to the latest Union Internationale Centre le Cancer criteria.19 Cases included 10 tumors in stage IA, 5 in stage IB, 1 in stage IIA, 3 in stage IIB, 10 in stage IIIA, 6 in stage IIIB, and 3 in stage IV (Table 1 ).


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  		Table 1. Histology and Stage of Lung Cancer in This Series of Patients*

 
Cells gathered from lung lesions were independently analyzed by conventional cytology and FISH. Informed consent for the cytologic examinations and genetic analyses of the specimens were obtained from all patients.

Cell Samples
In this study, the following four types of cell specimens were analyzed: cells obtained by TB (n = 18) and transbronchial fine-needle aspiration (n = 5) using a fiberoptic bronchoscope under fluoroscopy, CT scan-guided PN using the 19-gauge Tokyo Medical University Needle3 (n = 17), and BWs (n = 10). On every examination, duplicate specimens were made for simultaneous analyses of conventional cytology and FISH.

For conventional cytology, cells were stained by the Papanicolaou method. 5 Diagnosis was made by cytologists in the Department of Pathology at Tokyo Medical University Hospital. The various classes in conventional cytology are defined as follows: class I, absence of atypical or abnormal cells; class II, atypical cytology but no evidence of malignancy; class III, cytology suggestive of, but not conclusive for, malignancy (IIIa, mild dysplasia; IIIb, advanced dysplasia); class IV, cytology strongly suggestive of malignancy; and class V, cytology conclusive for malignancy.5

FISH
For FISH, cells on glass slides were air-dried overnight and stored at –80°C until they were used. Direct fluorochrome-labeled centromeric probes were used for the enumeration of different chromosomes. Spectrum-orange-labeled or Spectrum-green-labeled probes for the respective centromeric regions of chromosomes 3 and 17 were purchased (Vysis Inc; Downers Grove, IL), and dual-color FISH was performed. Slides were denatured by incubation with 70% formamide (two times the standard saline citrate [SSC] solution) at 74°C for 2 min in a water bath. Then, slides were dehydrated through a graded ethanol system (70% for 2 min, 85% for 2 min, and 100% for 2 min). A hybridization solution (10 µL) was applied to each slide, which was coverslipped and sealed with rubber cement. The hybridization solution contained 1 µL each DNA probe in 70% formamide (two times the SSC solution), and 10% dextran sulfate solution (cot I DNA). After incubation for 16 h at 37°C in a humidified chamber, slides were washed (two times SSC solution) for 3 min at 74°C. A di-amidinophenylindole antifade solution (8 µL) was applied to each spot and coverslipped. The slides were observed under a fluorescence microscope that was connected to a cooled charge-coupled device camera and an image analyzer system (CytoVision; Applied Imaging, Ltd; Newcastle, UK).

FISH signal analysis was performed as follows. All cells in a fluorescence microscopy field, except for those with damaged or overlapped nuclei, were evaluated. One hundred cells were counted, and the numbers of each centromeric signal were recorded. If there were < 100 cells on the slide, as many cells as possible were counted. When the percentages of hyperdisomic cells (ie, more than three copies for at least one chromosome) were > 10%, we judged the lesion to be malignant.

Comparison of Conventional Cytology and FISH
FISH diagnoses were made without clinical information or the results of conventional cytology. The results of FISH analysis were not shown to the cytologists. Thus, both diagnoses were independently made in a blind fashion.

Statistical Analysis
Differences in the number of countable cells according to the histology of the lung lesions or the cell-gathering methods used were analyzed by the Kruskal-Wallis test. A p value of < 0.05 was considered to be significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
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Cells countable for FISH analyses ranged from 5 to 100 (maximum). Cell counts according to the cell-gathering method did not differ significantly, but the fewest cells were obtained by PN (Fig 1 ). Although the fewest cells were obtained from small cell carcinomas, no significant difference was seen according to the histologic type of lung cancer (Fig 2 ).



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  		Figure 1. Countable cells according to the type of clinical specimen. Although cell counts obtained by PN were the lowest, no statistical significance was obtained by the Kruskal-Wallis test (p = 0.1117). Error bars indicate standard error. TN = transbronchial fine-needle aspiration.

 


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  		Figure 2. Countable cells according to histology. Although cell counts obtained from small cell lung cancer (Sm) were the lowest, no statistical significance was obtained by the Kruskal-Wallis test (p = 0.2369). Error bars indicate SE. Ad = adenocarcinoma; B = benign lesion; La = large cell carcinoma; RCC = renal cell carcinoma; Sq = squamous cell carcinoma.

 
The results of conventional cytology according to the Papanicolau classification were as follows: class I, 4 cases; class II, 15 cases; class IIIa, 3 cases; class IIIb, 5 cases; and class V, 23 cases (Table 2 ). Twenty-eight cases showing a higher grade than class IIIb were considered to be positive for lung cancer. By cytology, we found no false-positive cases and 11 false-negative cases. Thus, by conventional cytology, specificity was 100%, and sensitivity was 71.8%.


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  		Table 2. Results of FISH and Conventional Cytology*

 
In FISH analyses, 34 cases showed aberrant copy numbers in either chromosome 3 or 17. Representative findings of conventional cytology and FISH are shown in Figure 3 . We found no false-positive cases and five false-negative cases. For FISH analyses, specificity was 100%, and sensitivity was 87.1%.



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  		Figure 3. Hyperdisomic cells detected by FISH. Red signals are the centromeric region of chromosome 3, and green signals are the centromeric region of chromosome 17.Representative findings of conventional cytology (Papanicolaou stain, original x400) and FISH in the same cases, as follows: top left, A: adenocarcinoma (case 39); top right, B: squamous cell carcinoma (case 1); bottom left, C: large cell carcinoma (case 24); and bottom right, D: small cell carcinoma (case 11).

 
Seven cases, including one with metastatic renal cell carcinoma, had negative cytology findings and positive FISH findings. In these cases, the cytologic classifications were as follows: class II, five cases; and class IIIa, two cases. One case was cytology-positive and FISH-negative. In this case, the portion of aneusomic cells were observed to be 8 to 9%, which is just below the predetermined cutoff value.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated here the usefulness of FISH analyses in diagnosing lung cancer using various clinical specimens. Similar results about the effectiveness of FISH analyses have been reported by several authors. Schenk et al20 examined 23 patients with lung cancer by FISH with probes specific for chromosomes 3, 8, 11, 12, 17, and 18 for malignant effusions and primary tumors. In that study, chromosomal alterations always consisted of gains in chromosomal signal numbers, and all chromosomes were found to be aneusomic to a similar extent. According to this observation, we used only two probes in the present study, which were specific for chromosomes 3 and 17.

Recently, Sokolova et al21 analyzed BW specimens from 48 patients with lung cancer by FISH using four probes (ie, centromeric region of chromosome 1, 5p15, 8q24 (c-myc), and 7p12 [epidermal growth factor receptor]). In that report, FISH detected 15 of 18 specimens that were falsely negative by cytology. The sensitivity of FISH for the detection of lung cancer was 82% compared with 54% sensitivity by conventional cytology. The same group22 used a similar FISH probe set to show that significantly higher frequencies of abnormal cells were found in each of the 20 surgical specimens of non-small cell carcinoma (100%) and in the 3 sputum specimens (100%) from lung cancer patients. These probes detected a 4.8 to 7.3% rate of abnormal copy numbers in normal control specimens. In these retrospective studies, FISH detected lung cancer cells in touch preparations of resected tumors and BWs. Thus, we planned a prospective study to compare conventional cytology with FISH using various specimens from lung lesions.

In our study, we determined the cutoff value for the percentage ofhyperdisomic cells to be 10%, because we often count less double equals6% hyperdisomic cells in normal cell specimens, probably due to counting sister chromatids as two copies. When we set the cutoff value at 10%, a specificity of 100% and a sensitivity of 87.1% were obtained by FISH, whereas the sensitivity of cytology was 71.8%. As a result, we successfully detected seven lung cancer cases that were cytology-negative. Among these cases, two were class IIIa that we could not diagnose as malignant based on morphologic features. FISH may provide decisive information for the detection of malignancies, especially cases with IIIa classification.

Although the sensitivity of FISH is superior to that of conventional cytology, there are some disadvantages to FISH analyses. First, we do not generate information about the histologic type of lung cancer since we cannot observe morphologic features. Second, FISH is expensive. Third, FISH signal counting under fluorescence microscopy is time-consuming. Thus, the present FISH assay system probably can play a complementary role to that of conventional cytology.

We had five cases that we could not correctly diagnose by FISH. There are two possible reasons for our false-negative FISH results. One would be the failure to obtain proper cell material from the lesion, resulting in the absence of cancer cells on the slide. The other would be that the cancer cells were near-diploid, such that we could not detect aneusomy in two target chromosomes. We could probably detect more aneusomic cells using additional suitable probes for other chromosomes or chromosomal regions as reported by Romeo et al,22 who successfully diagnosed 100% of lung cancer cases by FISH using a set of four probes. In our previous study,23 chromosomal instability detected by FISH was associated with poor survival in patients with lung cancer. The finding of multiple chromosomal changes by FISH may be used as a prognostic factor and in the selection of patients for different therapeutic programs in the future.

In conclusion, FISH can detect lung cancer cells with aneusomy in various clinical specimens. The sensitivity was superior to that of conventional cytology. FISH should be used in conjunction with conventional cytology.


   Footnotes
 
Abbreviations: BW = bronchial washing; FISH = fluorescence in situ hybridization; PN = percutaneous needle biopsy; SSC = standard saline citrate; TB = transbronchial brushing

Supported by grant No. 15591493 from the Ministry of Education, Science, Sports, and Culture in Japan.

Received for publication September 2, 2004. Accepted for publication January 13, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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