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. Author manuscript; available in PMC: 2012 Jun 8.
Published in final edited form as: J Thorac Oncol. 2008 Sep;3(9):979–984. doi: 10.1097/JTO.0b013e3181834f3a

3p22.1 and 10q22.3 Deletions Detected by Fluorescence In Situ Hybridization (FISH)

A Potential New Tool for Early Detection of Non-small Cell Lung Cancer (NSCLC)

Sai Yendamuri *, Ara A Vaporciyan †, Tanweer Zaidi ‡, Lei Feng §, Ricardo Fernandez ‡, Nebiyou B Bekele §, Wayne L Hofstetter †, Feng Jiang ||, Reza J Mehran †, David C Rice †, Margaret R Spitz , Stephen G Swisher †, Garrett L Walsh †, Jack A Roth †, Ruth L Katz ‡
PMCID: PMC3370669  NIHMSID: NIHMS381558  PMID: 18758299

Abstract

Background

Our objective was to study the feasibility of detecting chromosomal deletions at 3p22.1 and 10q22.3 by fluorescent in situ hybridization (FISH) and to examine their distribution in different areas of the airway in patients with non-small cell lung cancer.

Methods

Brush biopsies from the mainstem bronchus on the normal side contralateral to the tumor (NBB) and mainstem bronchus on the tumor side (TBB) were obtained from 122 patients who underwent surgical resection. Touch preparations from the tumor (TTP), normal lung parenchyma, and bronchi adjacent to the tumor were also obtained. Two FISH assays using probes complementary to 3p22.1 and 10q22.3 were used to detect deletions.

Results

NBB showed a relatively low deletion rate of 3p22.1 and 10q22.3 compared with TTP (p < 0.0001). TBB showed a significantly higher rate of deletions compared with NBB but lower than TTP from the tumor (p < 0.05) for both 3p22.1 and 10q22.3. A significantly higher deletion rate was seen at TTP compared with normal lung parenchyma at both the 3p22.1 and 10 q22.3 (p < 0.0001). Correlations were seen between the deletion rates of TTP and TBB at 3p22.1 (ρ = 0.61, p < 0.0001) and between TTP and bronchi adjacent to the tumor at 10q22.3 (ρ = 0.64, p < 0.0001).

Conclusion

Deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISH. As one progresses from the contralateral normal bronchus to the bronchus on the side of tumor and the tumor itself, the percentage of chromosomal deletions increases in a statistically significant fashion. This suggests that, FISH analysis of bronchoscopic brushes may be useful for identifying patients at high risk for developing non-small cell lung cancer.

Keywords: Lung, Cancer, Non-small cell, Screening, Fluorescent in situ hybridization, Bronchial brushes

Lung cancer is estimated to affect 213,380 patients and result in 160,390 deaths in the United States in 2007 alone (www.lungusa.org), making it the most lethal cancer, both among men and women. Although early stage lung cancer is eminently curable by surgery,1 most lung cancer is detected at an advanced stage. This makes the overall outlook of lung cancer dismal. This underscores the importance of developing methods of early detection for lung cancer. Several approaches to early detection have been evaluated. High resolution computed tomography scanning is one such approach. Another approach is to use sputum cytology2 and various modes of bronchoscopy including white light bronchoscopy and autofluorescence bronchoscopy.3 These techniques have their own limitations. Bronchoscopy has limited utility in the detection of peripheral lesions (which are forming an increasing proportion of non small cell lung cancer [NSCLC]). Sputum cytology has a low sensitivity in the detection of lung cancer. One of the ways that the sensitivity of sputum cytology has been improved is to study genetic changes in bronchial cells seen in sputum that predate morphologic changes detected by cytology.4 Several methods have been used to detect early changes, including chromosomal changes. One of these ways is fluorescent in situ hybridization (FISH) technology.5,6

FISH uses segments of DNA labeled with a fluorescent dye that can be detected at prespecified wavelengths. These segments of DNA are hybridized to cells on slides and compared with their corresponding centromeric region to detect gain or loss of the chromosomal segment in question. This enables the detection of a few abnormal cells in a background of predominantly normal cells, which is typically found in sputum cytologic specimens. This method has been found to be useful in bladder cancer. A similar set of probes has been used in the detection of abnormal cells in sputum cytology based on the detection of chromosomal gain in the 5p, 7p, and 8q regions7 These probes have been shown to improve the sensitivity of sputum cytology. We have developed in-house probes for the detection of chromosomal deletions at the 3p22.1 and 10q22.3 regions. These regions have been previously shown to be chromosomally deleted by CGH array data.8 In this study, these probes have been used to measure the deletion rate of these chromosomal regions in bronchoscopic brush biopsies. We show that this technique is feasible and shows instructive data on the distribution of these deletions.

PATIENTS AND METHODS

Patient Population and Specimen Collection

Patients presenting to the thoracic surgery clinic at our institution for potential resection were consented for acquisition of bronchoscopic brush biopsies according to an IRB approved protocol. Patients were entered into the study only if they were deemed resectable and did not receive any preoperative radiation or chemotherapy. Biopsies were performed at the time of their preoperative bronchoscopy immediately before the proposed resection. Clinical and pathologic data was extracted from the prospective thoracic surgery database maintained in our institution. Brush biopsies were obtained from bronchoscopically normal mainstem bronchus both on the side of the lesion (TBB) and the opposite side (NBB). Once the tumor was resected and sent for pathologic analysis, touch preps were made from bronchus adjacent to the tumor (TAB), from the tumor itself (TTP) and from normal lung parenchyma away from the tumor (NTP), depending on the amount of tissue remaining after clinical pathologic use (Figure 1).

FIGURE 1.

FIGURE 1

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Schematic showing the areas of biopsy. NBB, brush biopsy of the mainstem bronchus on the side opposite the tumor; TBB, brush biopsy of the mainstem bronchus on the side of the tumor; TAB, touch prep of the bronchus adjacent to the tumor; NTP, touch prep of normal lung parenchyma; TTP, touch prep of the tumor.

Specimen Preparation

The brush biopsies were received in saline. Cytospins were prepared for cytology and FISH analysis. The cytology slides were fixed in Carnoy’s solution and stained with Papanicolaou stain, whereas the air dried slides were stained with Diff-Quik (Baxter Scientific, Deerfield, IL). The cytologic features of all the specimens were assessed. Both the cytospins and the touch imprints were fixed for FISH in methanol and acetic acid at 3:1 ratio before labeling. FISH was performed with two probe sets.

FISH Technique

Two probe sets were used. The first probe set included centromeric 3 (CEP3; Vysis) and the locus specific probe 3p22.1, which was labeled in-house. The second probe set consisted of centromeric 10 (CEP10; Vysis) and the locus specific probe 10q22.3 which was also developed in-house. These probes were mixed with blocking DNA, enriched in repetitive sequences and hybridized to the specimens. Spectrum Green was used to label the locus specific probes while the centromeric probes came labeled with Spectrum Orange.

The technique for making the probes has been previously described6,9. Briefly, DNA was labeled by a commercial nick translation kit. A gel was run to confirm that the size of the DNA fragments was 500 to 2000 base pairs. The probe was then placed in a 65°C water bath for 15 minutes and then stored at −20°C.

Slides were immersed in 2X standard sodium citrate (SSC) for 3 minutes at 77°C and then in a protease solution (50 mL of 1 X phosphate-buffered saline [PBS], pH 2.0, and 25 mg of protease). Protease digestion was then performed (5– 6 minutes for cytospins and7– 8 minutes for touch preparations). Slides were then washed in 1 X PBS for 5 minutes and subsequently fixed in a 1% formaldehyde solution for 5 minutes. Later, the slides were again washed with 1 X PBS for 5 minutes and dehydrated in sequential 70%, 85%, and 100% ethanol solutions. After a brief period of drying, the probe mixture was applied to the target areas on each slide, covered with a cover slip, and sealed with rubber cement. The slides were then kept in a hybridization machine (Vysis) for ≥20 hours at 37°C. After the rubber cement and cover slips were removed, the slides were washed in a 50% formamide solution (pH 7.45) in 3 separate jars for 10 minutes each at 45°C. Next, the slides were washed in 2 X SSC for 10 minutes at 45°C and then in 2 X SSC/0.1% ethoxylated octyl phenol (NP-40) for 10 minutes. The slides were then dried. Slides were counterstained with 4′ 6-Diamidine-2-phenylin-dole dihydrochloride (10 μl per slide), cover slipped, and viewed under a fluorescent microscope (Leica DWRXA or Leica DMLB; Leica Microsystems, Inc., Buffalo, NY) with the appropriate filters for the probes.

The hybridized slides were counted with the appropriate filter sets for visualizing Spectrum Green or Spectrum Orange and 4′ 6-Diamidine-2-phenylindole dihydrochloride counterstain. The nuclei of individual cells that did not overlap were chosen for analysis. Slides were analyzed only if 80% of the cells were interpretable in the field of view and the brightness of the signals was >2+ on a scale of 0 to 3+. Each cell was scored individually for the number of green (3p22.1 or 10q22.3) and orange signals (CEP3 or CEP10, respectively) that were used as internal controls. Cells with fewer green signals than orange signals were positive, reflecting deletion. The ratio of green to orange signals was interpreted as the percentage of deleted cells. To avoid misinterpretation due to insufficient hybridization, cells were counted only if at least one bright orange and one bright green signal were present. Split signals were counted as one if the space between them was less than the diameter of a single signal. A control specimen was used for each batch. Figure 2 shows representative slides of the FISH technique used. From the NBB, TBB, TAB, and NTP sites, deletions were scored in 100 morphologically intact bronchial epithelial cells at each site. In tumor touch preparations, at least 100 tumor cells were evaluated for deletions of 3p22.1 and 10q22.3 relative to the internal centromeric probes. The accuracy of manual scoring was confirmed by a random sample check performed independently by a second cytogenetic technologist. This score showed a strong correlation with the first score.

FIGURE 2.

FIGURE 2

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Sample slides of the FISH analysis. Orange signals are centromeric signals. Green signals indicate 3p22.1 and 10q22.3 probes. The cells with fewer green signals than orange signals show cells that have deletions at either the 3p22.1 (A) and 10q22.3 (B) loci.

Analysis of Data

Deletions rates at different locations were tabulated in percentages. To compare the deletion rates at different sites, these were normalized to their technical controls before comparison. The Wilcoxon signed rank test was used for comparison of deletion rates at different sites. Correlations of FISH measurements between the brush biopsies (TBB and TAB) and the tumor touch preps (TTP) were evaluated by Spearman correlation coefficients. Kaplan-Meier curves were plotted for estimating time to death distributions. The log-rank test was used to compare the difference in survival and recurrence free survival between the FISH measurement high score and low score groups, defined by the median deletion rate. All tests are two-sided, and p-values less than 0.05 are considered statistically significant. Analyses were performed using the SPSS software version 13.0 (SPSS Inc., Chicago, IL).

RESULTS

Table 1 shows the demographic and clinical data that describes this cohort of 122 patients included in the study. The high percentage of women (52.5%) and adenocarcinomatous histology (56.5%) is reflective of changing epidemiologic trends in NSCLC. Very few of these patients have well differentiated tumors (9.8%) and most patients are in the earlier stages of the disease, as would be expected in a patient group deemed resectable without neoadjuvant therapy.

TABLE 1.

Clinical Characteristics of the Study Population

Demographics n = 122
Age
 Mean 66
 Range 32–84
Gender
 Male 47.5%
 Female 52.5%
Histology
 Adenocarcinoma 69 (56.5%)
 Squamous cell cancer 37 (30.3%)
 Adenosquamous 3 (2.4%)
 NSCLC (not specified) 7 (5.8%)
 Other neoplasm 6 (4.8%)
Grade
 Well differentiated 12 (9.8%)
 Moderately differentiated 52 (42.6%)
 Poorly/undifferentiated 49 (40.2%)
 Grade not specified 9 (7.2%)
Pathologic stage
 IA 29 (23.8%)
 IB 40 (32.8%)
 IIA 3 (2.5%)
 IIB 13 (10.7%)
 IIIA 21 (17.2%)
 IIIB 10 (8.2%)
 IV 6 (4.9%)
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Table 2 demonstrates the success rate of data acquisition at different locations. Although a high percentage of the bronchial brushes could be analyzed successfully with FISH, lower numbers of tumor and normal lung parenchyma could be analyzed. A large part of this difference is attributable to the sequence of specimen acquisition. Although bronchial brushes were obtained on all patients first, a subset of these patients underwent mediastinoscopy before surgical resection. If N2 or N3 nodes were positive at mediastinoscopy, surgical resection was abandoned at the time, in keeping with current practice at our institution. Specimens at TTP, NTP, and TAB could therefore not be obtained from these patients. Furthermore, some patients did not have an area suitable for obtaining TAB specimens after clinical pathologic use, further limiting the specimens available from this area. The mean deletion rate at different locations for both 3p22.1 and 10q22.3 are also tabulated here. The highest deletion rate is obtained from TTP at both loci. The next highest deletion rate is obtained from bronchi adjacent to the tumor (TAB) or on the same side (TBB). The lowest deletion rate is from areas farthest away from the tumor which include normal lung parenchyma (NTP) and mainstem bronchi on the nontumor side (NBB) (Figure 3).

TABLE 2.

Normalized Deletion Rate Detected by FISH in Different Regions of the Airway

3p22.1 N % Mean Deletion Rate ± SE 10q22.3 N % Mean Deletion Rate ± SE
3p22.1NBB 119 97.5 1.14 ± 0.12 10q22.3NBB 119 97.5 0.53 ± 0.06
3p22.1TBB 120 98.4 2.80 ± 0.26 10q22.3TBB 119 97.5 1.61 ± 0.09
3p22.1TAB 70 57.4 1.93 ± 0.2 10q22.3TAB 69 56.6 1.76 ± 0.15
3p22.1NTP 95 77.9 1.46 ± 0.14 10q22.3NTP 93 76.2 0.88 ± 0.09
3p22.1TTP 96 78.7 7.83 ± 0.59 10q22.3TTP 96 78 4.76 ± 0.34
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FIGURE 3.

FIGURE 3

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The deletion rate at 3p22.1 and 10q22.3 increases systematically as one progresses from tissue away from the tumor towards the tumor itself. The deletion rate is significantly greater in brush biopsies from mainstem bronchi on the tumor side (TBB) compared with the normal side (NBB) (p < 0.05). Similarly, the deletion rate at the tumor itself (TTP) is significantly greater than TBB itself (p < 0.05).

The deletion rate at different areas was compared with the deletion rate at TTP using the Wilcoxon signed rank test. The deletion rate at TTP was statistically significantly higher than each of the other areas. There is also a statistically significant difference in the deletion rate between the main-stem bronchi on the tumor side and the nontumor side (TBB and NBB). This holds true at both loci- 3p22.1 and 10q22.3.

Correlations between areas with a low deletion rate and TTP were also determined. A strong statistically significant correlation is demonstrated between the deletion rate at TBB and the deletion rate at TTP at the 3p22.1 locus (Figure 4A; spearman correlation coefficient = 0.61). A similar statistically significant correlation is seen between the deletion rate at TAB and the deletion rate at TTP at the 10q22.3 locus (Figure 4B; spearman correlation coefficient = 0.64). The deletion rate of 3p22.1 at different locations was not predictive of survival (data not shown). The median deletion rate at 10q22.3 both at TBB and TTP separated two groups that demonstrated differing survival trends. This difference was not statistically significant (Figure 5).

FIGURE 4.

FIGURE 4

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A, 3p22.1 deletions at TBB correlate with 3p22.1 deletions at TTP. Spearman coeff = 0.61, p < 0.00001. B, 10q22.3 deletions at TAB correlate with 10q22.3 deletions at TTP. Spearman coeff = 0.64, p < 0.00001.

FIGURE 5.

FIGURE 5

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Survival curves for patients separated by median deletion rate at the 10 q locus at (A) TTP (p = 0.176) (B) TBB (p = 0.153).

DISCUSSION

Early detection of lung cancer is of paramount importance due to the vast gap between the overall survival of patients diagnosed with lung cancer and the survival of patients with early lung cancer.10 Improvement in imaging has renewed interest in image based screening and early diagnostic efforts11 after earlier studies using chest radiograph screening were negative, presumably due to low sensitivity of the screening test.12 Similarly, methodological improvement in the detection of chromosomal alterations in sputum and bronchoscopic specimens has led to a renewed interest in their use in the screening of lung cancer. Sputum and bronchoscopic cytologic analysis has been limited by low sensitivity. Fluorescent in situ hybridization increases the sensitivity of cytologic analysis13 as this technique is better at the detection of a small number of abnormal cells in a background of normal cells.

A number of molecular changes have been described in lung cancer. These include both loss of tumor suppressor genes and activation of oncogenes.14,15 The 3p region is presumed to be the site of several tumor suppressor genes including FHIT, RASSF1A, and FUS1.16 –18 It is one of the earliest genetic losses in lung tumorigenesis. Therefore, using a probe in this region is a logical choice for early detection. The 10q22.3 region that is used as a probe in our study includes the gene encoding the surfactant associated protein A (SP-A). Pulmonary surfactant is important for normal lung physiology and alterations in surfactant have been associated with a number of lung diseases in children and adults. Of the 4 surfactant proteins, SP-A is the most abundant. The SP-A gene locus consists of 2 homologous genes, each about 4.5 kb in length separated by an 59 kb area.19 Alterations in SP-A have been looked at using various techniques including reverse transcription-polymerase chain reaction, immunohistochemistry, immunoblot analysis, and enzyme-linked immunosorbent assay, with inconsistent results.20,21 In a cDNA microarray analysis, we have shown this area to be one of the more commonly deleted regions in lung cancer.8 Loss of this region has also been demonstrated to portend a poorer prognosis in patients with early stage lung cancer.22

A number of previous studies have used FISH detected chromosomal gain but not loss to detect abnormal cells to improve detection.5,7,13 A pilot study performed by our group demonstrated the utility of FISH detected chromosomal loss in the diagnosis of lung cancer.9 Here, we present results of FISH detected deletions at the 3p22.1 and 10q22.3 locus in a larger cohort of patients.

From the data presented, several conclusions can be reached. The first is that FISH analysis with these probes is technically feasible and reproducible enough for routine clinical application. Good quality results can be obtained from minimally invasive bronchoscopic methods like bronchial brushes. The second conclusion is that there is a field effect that can be demonstrated in a quantifiable fashion. The pattern of this effect is intuitive, with the greatest deletion rate being seen in tissue closest to the tumor and the lowest deletion rate seen in tissue farthest away from the tumor. This pattern is easily recognized when comparing the deletion rate between the mainstem bronchus on the normal side (NBB) and the tumor side (TBB), raising the possibility of utilizing this data in the early detection of lesions. Of note, it is not necessary for the tumor to have squamous histology or be a central tumor to exhibit these changes. The strong correlation of deletion rates between the bronchoscopic brushes and the TTP not only increases the biologic validity of the data, but also raises the possibility of using the deletion rates in bronchoscopic specimens in lieu of TTP. This is particularly important given the trend in survival shown by patients with low deletion rates compared with patients with high deletion rates at TBB and TTP. This suggests that we can potentially use bronchoscopically acquired deletion rates for prognostication. The difference in survival in these groups corresponds well with earlier published data of the ability of SP-A expression to separate out patients with a good prognosis from patients with a bad prognosis.22 This data also raises the possibility of studying these deletions in sputum samples and this has been a focus of further investigation by our group.23

CONCLUSION

Chromosomal deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISH in brush biopsies and touch preparations. As one progresses from the contralateral normal bronchus to the bronchus on the side of tumor and the tumor itself, the percentage of chromosomal deletions increases in a statistically significant fashion. This suggests that FISH analysis of bronchoscopic brushes may be useful for identifying patients at high risk for developing NSCLC.

Acknowledgments

Supported by Specialized Program of Research Excellence in Lung Cancer grant P50CA70907, National Cancer Institute, Bethesda, MD-Roth, Spitz, Katz; (1) National Cancer Institute, NIH, Department of Health and Human Services (to M.R.S.) grant CA 55769; (2) M. Keck Center for Gene Therapy Award (to F.J.); (3) U.T. M. D. Anderson Cancer Center Institutional Research Grant (to F.J.); (4) U.T. M. D. Anderson Cancer Center Tobacco Settlement Fund (to F.J.).

The authors would like to acknowledge the assistance of Jinping Zhang and Hua-Zhong Zhang in collection of the data.

Footnotes

Disclosure: Drs. Katz and Jiang are holders of an issued USA patent (US Patent Appl. No. 20060078885) for FISH probes to 3p22.1 and 10q22-23. The other authors declare no conflict of interest.

Presented at the 12th World Conference on Lung Cancer, Seoul, South Korea, September 2– 6, 2007.

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