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Clinical Cancer Research Vol. 9, 2294-2299, June 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Chromosomal Instability Detected by Fluorescence in Situ Hybridization in Surgical Specimens of Non-Small Cell Lung Cancer Is Associated with Poor Survival1

Haruhiko Nakamura2, Hisashi Saji, Aute Idiris, Norihito Kawasaki, Makoto Hosaka, Akihiko Ogata, Takamoto Saijo and Harubumi Kato

Department of Surgery, Tokyo Medical University Hospital, Tokyo 160-0023, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Chromosomal instability (CIN) in non-small cell lungcancer (NSCLC) has yet to be well studied. We examined the relationship between CIN detected by fluorescence in situ hybridization and survival in patients with NSCLC.

Experimental Design: Touch preparations from 50 surgical specimens of NSCLC were studied. Tumors included 34 adenocarcinomas, 15 squamous cell carcinomas, and 1 large cell carcinoma. The pathologic stage was IA in 14, IB in 17, IIB in 8, IIIA in 9, and IIIB in 2 cases. Enumeration of chromosomes 3, 10, 11, and 17 was used to determine which tumors carried CIN. The association between CIN and survival was also analyzed.

Results: Disomy was most common, but tetrasomy and trisomy of the examined chromosomes were seen frequently. Fourteen tumors (28%) showed heterogeneity of all four chromosomes examined and were judged to be carrying CIN. Both univariate and multivariate analyses revealed that two factors, lymph node metastasis and CIN, were significant poor prognostic factors.

Conclusions: CIN in NSCLC detected by fluorescence in situ hybridization is an independent factor predicting a poor prognosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genetic instability in cancer cells includes two factors, MIN3 and CIN. MIN is observed at the nucleotide level and results in base substitutions or deletions or insertions of a few nucleotides. On the other hand, CIN occurs at the chromosomal level, resulting in losses and gains of whole chromosomes (1) . CIN was identified using FISH analysis to count the specific fluorescence signals from targeted chromosomes in the interphase nuclei of colorectal cancer cell lines (1) . The cell lines examined in that report were clearly classified into two groups: (a) near diploid cell lines with MIN and (b) aneuploid cell lines with CIN. On the basis of these findings, primary colorectal cancer progression from near diploid to aneuploid tumors was demonstrated, probably along a pathway of multistep carcinogenesis, including mutations of various oncogenes or tumor suppressor genes (2) . In that study, aneuploid colon cancers, as defined based on DNA content measured by laser scanning cytometry, showed intercellular numerical variation in chromosomes 7, 17, and 18 by FISH analysis. Although aneuploidy is a common feature of many different solid tumors, aneuploidy does not always imply the existence of CIN. Because CIN generates intercellular numerical variation in the same chromosomes within a given tumor, FISH analysis is a practical method to detect CIN in surgical specimens (2) .

In lung cancer, MIN is probably a function of the inactivation of mutation repair genes and less common than in cancers of the digestive tract (3, 4, 5) . Therefore, CIN might play a more important role than MIN in lung cancer progression. Although aneuploidy in lung cancer is common (6) , CIN has not been well studied in clinical specimens. We hypothesized that NSCLCs with CIN probably have aggressive phenotypes, because numerous genetic abnormalities worsen the prognosis. We first examined whether or not CIN is present in NSCLC. Second, we analyzed the relationship between CIN and survival. As far as we know, the present study is the first analysis of CIN in NSCLC detected by FISH and also the first to examine its impact on survival.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
Surgical specimens from 50 patients with NSCLC who underwent resection in our hospital from December 1997 to December 1999 were studied. The patients included 27 men and 23 women, with an average age of 67 years. Lobectomy and mediastinal lymph node dissection were performed in all cases. No presurgical chemotherapy or radiotherapy was administered. The diagnosis of NSCLC was established by histological examination of the surgical specimens. Tumors included 34 adenocarcinomas, 15 squamous cell carcinomas, and 1 large cell carcinoma. Tumor-Node-Metastasis staging of the cancers was performed using the latest criteria (7) . The pathologic stage was IA in 14, IB in 17, IIB in 8, IIIA in 9, and IIIB in 2 cases. Informed consent for genetic analysis of the primary lung cancer was obtained from all patients. All patients have been followed in the outpatient department of our hospital for a mean of 43.9 months after resection of the tumor.

FISH.
Touch preparations were made immediately after resection of the tumor by touching the fresh cut surface of the primary tumor to a glass microscopy slide. Specimens were air dried and stored at -20° until use. Direct fluorochrome-labeled centromeric probes were used for enumeration of different chromosomes. Spectrum orange-labeled or Spectrum green-labeled probes for the centromeric region of chromosomes 3, 10, 11, and 17 were purchased from the manufacturer (Vysis, Inc., Downers Grove, IL). In brief, the slides were denatured by incubation with formamide (70% in 2 x SSC) at 74° for 2 min in a water bath. Then, the slides were dehydrated through a graded ethanol system (70% for 2 min, 85% for 2 min, and 100% for 2 min). Hybridization solution (10 µl) was applied to each slide, which was coverslipped and sealed with rubber cement. The hybridization solution contained 1 µl of the DNA probe in 70% formamide, 2 x SSC, and dextran sulfate (10%, cot I DNA). After incubation for 16 h at 37° in a humidified chamber, slides were washed with 2 x SSC for 3 min at 74°. Then, 4',6-diamidino-2-phenylindole-antifade solution (8 µl) was applied to each spot and coverslipped. The slides were observed under a fluorescence microscope connected to a cooled charge coupled device camera and an image analyzer system, CytoVision (Applied Imaging, Ltd., Newcastle, United Kingdom).

FISH signal analysis was performed as follows. All cells in a fluorescence microscopy field, except those with typical morphological features of granulocytes or lymphocytes or damaged or overlapped nuclei, were evaluated. One-hundred cells were counted, and the numbers of each centromeric signal were recorded. CIN was defined as present when the sum of the percentages of cell nuclei that do not carry a modal copy number of each chromosome was >25% in all four examined chromosomes. This cutoff value of 25% was determined based on a previous report in which all colorectal cancer cell lines without CIN had <25% of cells with a nonmodal chromosome number (1) . We called the percentage of cells that do not carry the modal copy number the "CIN index."

Statistical Analysis.
The survival rate was calculated using the Kaplan-Meier method. The significance of survival differences between subgroups was tested by the Log-rank method as a univariate analysis. A multivariate survival analysis was performed using the Cox proportional hazards model. A model obtained with step-down variable selection, in which all prognostic factors were initially entered into the model and in which nonsignificant factors (P > 0.05) were rejected, was compared with the primary model, which included all prognostic factors, regardless of their measured significance. The association between the two factors was tested by the {chi}2 test. A result was considered significant when the P was <0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The copy number of chromosomes 3, 10, 11, and 17 was analyzed in 50 primary NSCLCs. The modal copy number of each chromosome ranged from two copies (disomy) to six copies (hexasomy), and the percentage of cells that did not carry the modal copy number was quite variable (Table 1)Citation . In addition to disomy, tetrasomy and trisomy were common (Table 2)Citation . Representative FISH findings in cancer cells carrying aneusomy of chromosome 11 (modal copy number = three) without numerical heterogeneity and cancer cells carrying aneusomy of chromosome 3 with numerical heterogeneity are shown in Fig. 1Citation .


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  		Table 1 Modal copy number and instability of chromosomes in NSCLC detected using FISH

 

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  		Table 2 The prevalence of aneusomy in surgical specimens of NSCLC

 


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  		Fig. 1. Representative FISH analysis. A, a tumor in which most cancer cells carry three copies of the centrometric signal of chromosome 11 (green dots). B, a tumor in which cancer cells are heterogeneous for the copy number of the centrometric signal of chromosome 3 (red dots).

 
Fourteen tumors (28%) showing numerical heterogeneity in all four examined chromosomes were judged to be carrying CIN. A representative distribution of the chromosome copy numbers in a tumor with CIN and tumor without CIN are shown in Fig. 2Citation . Some tumors showed numerical heterogeneity of some chromosomes, but we did not consider this type of abnormality as CIN in this study, because CIN should change chromosome numbers at the genome-wide level.



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  		Fig. 2. Representative distributions of the chromosomal copy number. In A, a tumor without CIN is homogeneous for the copy number of each chromosome. In B, a tumor with CIN shows a heterogeneous numerical distribution for each chromosome.

 
The association between patient survivals and clinical factors, including CIN, was analyzed by univariate and multivariate analyses (Table 3)Citation . Log-rank test revealed that two factors, lymph node metastasis and CIN, predicted a poor outcome (Fig. 3)Citation . On multivariate analysis, both regional lymph node metastasis and CIN were significant poor prognostic factors. Possible associations between the presence of CIN and other potential prognostic factors (gender, age, T factor, N factor, presence of microscopic vascular and lymphatic invasion, and differentiation grade) were examined by the {chi}2 test, and none were identified. Thus, CIN detected by FISH was considered as an independent poor prognostic factor in NSCLC.


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  		Table 3 Prognostic factors in patients with NSCLC

 


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  		Fig. 3. Kaplan-Meier survival curves after resection of NSCLC. Lymph node metastases and with CIN positivity was associated with a poor prognosis. A, survival according to status of lymph node metastasis. B, survival according to CIN status.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Aneuploidy is a characteristic feature of many solid tumors, including lung cancer (8) . Recently, an association between aneuploidy, defined by DNA content, and prognosis of patients with NSCLC was subjected to a meta-analysis (9) . That report analyzed 35 studies and concluded that patients with nearly diploid tumors benefited from a significant reduction in the risk of death at 1, 2, 3, and 4 years, with a respective odds ratio of 0.51, 0.51, 0.45, and 0.67. Although results are conflicting (10) , aneuploidy is probably a worse prognostic factor.

Aneusomy is common in a variety of cultured cancer cell lines derived from different organs (11) . However, in primary cancers, it is difficult to count the number of chromosomes, because karyotyping is impossible without short-term culture to obtain a good metaphase specimen. FISH is a convenient way to count numbers of specific chromosomes in interphase nuclei (12) . The classic cytogenetic analysis of NSCLC used karyotyping of short-term cultured cells and revealed various numerical chromosomal abnormalities (13) . Polysomy was common, as we reported here. We found that CIN exists in NSCLC and that it correlated with survival using FISH analysis.

We believe that the presence of CIN has a greater impact on survival than aneuploidy itself. Although tumor cells carrying CIN have aberrations in mitotic checkpoint genes, such as hBUB1, hsMAD2, and hBUBR1, frequent mutations in these loci have not been found in lung cancer (14, 15, 16) . Because the genes that are responsible for CIN in lung cancer have not yet been identified, the most practical way to detect CIN in primary cancer might be by direct numerical analysis of chromosomes.

Aneusomy of at least one of the chromosomes we examined was observed in 71.4% (10 of 14) of stage Ia NSCLCs. Therefore, polysomy appears to be a relatively early event in carcinogenesis. We are planning to confirm this hypothesis using specimens of NSCLCs in the earliest stages, such as atypical adenomatous hyperplasia and carcinoma in situ of the bronchial epithelium.

The limitations of our study are as follows. In counting FISH signals, it is difficult to exclude contamination by normal stromal cells, and only a limited number of chromosomes were analyzed. However, considering the results of previous studies on CIN and of FISH analyses of other cancers (1 , 2 , 17) , counting all chromosomes may not be necessary, because CIN probably induces numerical heterogeneity of most chromosomes. In the present study, instead of four chromosomes, heterogeneity of an individual single chromosome, 10, 11, and 17 might prove to be a prognostic factor on multivariate analysis, consequently. We found that some NSCLCs showed numerical heterogeneity in a limited number of chromosomes. We had one tumor with two unstable chromosomes and 10 tumors with one unstable chromosome. This phenomenon was not observed in a study on cell lines (1) but has been described in primary colon cancers (2) . Twenty-five lesions carrying at least one numerically heterogeneous chromosome had a worse prognosis in univariate analysis than tumors without heterogeneity. Thus, FISH analysis on one representative chromosome might be useful in predicting outcome in patients with NSCLC. If we define CIN using a CIN index of ≥10% for all 4 chromosomes, 19 cases (38%) are judged as CIN. But, under this criterion, the survival difference between patients with CIN and non-CIN tumors was not significantly different. Therefore, we used a CIN index of ≥25%, because it more reliably predicted survival.

CIN may be induced step by step, affecting individual chromosomes differently. Additional studies are needed on this point. The reason why the prognosis is worse when CIN is present is probably because of the accumulation of genetic alterations induced by CIN. Most patients with CIN died of distant disease. Gene expression microarray studies have shown that altered expression of various genes is associated with a significantly worse prognosis (18 , 19) , e.g., increased expression of ornithine decarboxylase, citron, thyroid transcription factor, and hepsin has been associated with favorable prognosis in adenocarcinomas (19) . Conversely, increased expression of ataxia-telangiectasia D-associated, prostaglandin E synthase, cathepsin L, and vascular endothelial growth factor C suggests a poor prognosis. Lung cancer cells with these malignant phenotypes might easily metastasize. However, additional data on the relationship between CIN and changes in the expression of individual cancer-related genes are necessary.

In conclusion, CIN can be detectable in primary NSCLC using FISH analysis. Because CIN was strongly associated with a worse prognosis by univariate and multivariate analysis, CIN can be considered an independent poor prognostic factor in NSCLC.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant 11671341 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Surgery, Tokyo Medical University Hospital, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. Phone: 81-3-3342-6111, extension 5071, 5838, 5845; Fax: 81-3-3342-6154; E-mail: hanakamu{at}tokyo-med.ac.jp Back

3 The abbreviations used are: MIN, microsatellite instability; CIN, chromosomal instability; NSCLC, non-small cell lung cancer; FISH, fluorescence in situ hybridization. Back

Received 11/20/03; revised 1/27/03; accepted 1/29/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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