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Chromosome 19 Translocation, Overexpression of Notch3, and Human Lung Cancer

Thao P. Dang, Adi F. Gazdar, Arvind K. Virmani, Tanya Sepetavec, Kenneth R. Hande, John D. Minna, John R. Roberts, David P. Carbone

Affiliations of authors: T. P. Dang, T. Sepetavec, K. R. Hande, D. P. Carbone, Division of Hematology and Oncology, Experimental Therapeutics Program, Vanderbilt-Ingram Cancer Center, Nashville, TN; A. F. Gazdar, A. K. Virmani, J. D. Minna, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas; J. R. Roberts, Division of General Thoracic Surgery, Vanderbilt-Ingram Cancer Center, Nashville.

Correspondence to: David P. Carbone, M.D., Ph.D., Division of Hematology and Oncology, Experimental Therapeutics Program, Vanderbilt-Ingram Cancer Center, 648 Medical Research Bldg. II, Nashville, TN 37232-6838 (e-mail: d.carbone{at}vanderbilt.edu).

Somatically acquired chromosome translocations are the major mechanism for gene activation in hematologic malignancies, but they have not been described previously in the more common epithelial tumors, such as lung cancers. Furthermore, lung carcinomas often exhibit complex karyotypes, and this complexity has been assumed to imply a lack of specificity. Indeed, chromosomal deletions associated with mutations in tumor suppressor genes, or gene amplifications of dominant oncogenes, are well-known genetic abnormalities in these tumors (1).

Here we describe the identification and mapping of a novel balanced t(15;19) somatically acquired translocation arising in an aggressive, metastatic lung carcinoma. A review of the literature shows three case reports (24) of t(15;19) translocations in undifferentiated epithelial intrathoracic tumors, diagnosed as thymic and/or lung in origin on the basis of the site of the disease and immunohistochemical findings. We have mapped the breakpoint to the 5` region of the highly overexpressed Notch3 gene and found its overexpression to be associated with karyotypic abnormalities of chromosome 19 in a panel of lung cancer cell lines. Notch3 is one of four known mammalian homologues of the Drosophila Notch receptor, essential for determining cell fate [for a review, see (5)]. Another Notch family member, Notch1, is involved in the t(7;9) translocation-associated acute T-cell lymphoblastic leukemia and is found to be overexpressed in cervical and colon carcinomas (6,7). Although mutations in the ligand-binding domain of Notch3 result in the CADASIL (i.e., cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) syndrome, Notch3, to our knowledge, has never before been implicated in human cancer (8).

The t(15;19) translocation was initially identified in an aggressive lung cancer metastatic to mediastinum and bone arising in a 34-year-old woman without a history of smoking or a family history of cancer. (All tissue and blood were collected under institutional review board-approved protocols with informed consent from the patient.) The tumor was karyotyped as 46,XX,t(15;19)(q11;p13) (Fig. 1Go, a). Extensive immunohistochemical studies (with positive staining for AE1/AE3 and CK7 and negative staining for leukocyte common antigen, {alpha}-fetoprotein, and S-100) indicated epithelial differentiation, and electron microscopy supported the diagnosis of poorly differentiated lung cancer. The karyotype of the peripheral blood of this patient was normal, indicating that this translocation was a somatic event, and a rapidly growing cell line derived from this tumor (HCC2429) was found to maintain this translocation stably after 1 year in culture (data not shown). The remarkable simplicity of the tumor karyotype and the stability of the translocation in this cell line suggested that this translocation identified a potent transforming activity in a chromosome region not known to harbor oncogenes important in lung cancer.



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Fig. 1. A balanced translocation involving 19p and 15q in non-small-cell lung cancer. Panel a: Karyotypic analysis from the original 2429 tumor specimen obtained at diagnosis, demonstrating the translocation involving chromosomes 15 and 19 [46,XX,t(15;19)(q11;p13)]. No other changes were seen. The chromosome breakpoints are indicated by arrows. Panel b: Fluorescence in situ hybridization analysis on the metaphase-arrested HCC2429 cell line with the use of the R31546 cosmid probe (B) and chromosome 15 {alpha}-satellite (A) for identification. Cosmid R31546 hybridizes to der(15), der(19), and chromosome 19, indicating that it spans the breakpoint. Panel c: Physical map of chromosome 19p, showing that the breakpoint was localized to cosmid R31546 by ordering the translocation in relationship to cosmid positions; asterisk (*) denotes translocation of cosmid probes to chromosome 15. Cosmid clones from chromosome 19 were obtained through a collaboration with the Human Genome Center at Lawrence Livermore National Laboratory. A chromosome 19 contig was available online at <http://www-bio.llnl.gov/bbrp/genome/genome.html>. Panel d: Northern blot analysis of lung cancer cell lines with chromosome 19 abnormalities by karyotypic analysis, showing Notch3 expression in HCC2429, NCI-H1819, H1793, H647, H513, and H441. All cell line designations beginning with the letter "H" and followed by numbers are NCI-H lines. Of note, all of the Notch3-expressing cell lines are from adenocarcinomas except for H513, which was from a mesothelioma. The lymphoblastoid cell line (BL2429) of the patient with t(15;19) translocation is shown in lane 1. Cyclophillin was used as loading control. NCI-H676, which also overexpresses Notch3, is not shown in this northern blot analysis.

 
Because of their limited resolution, traditional cytogenetic examinations often cannot identify loss of genetic material. We have studied the HCC2429 cell line for loss of heterozygosity (LOH) at 32 polymorphic markers (28 of which were informative) from chromosomes 19p and 15q. No LOH was observed on 19p or 15q when compared with the Epstein-Barr virus-transformed lymphoblastoid cell line (BL2429) established from the peripheral blood of the same patient (data not shown). These data indicated that there was no large region of deletion around the identified location of the translocation.

We mapped the translocation breakpoint by using fluorescence in situ hybridization (FISH) on metaphase-arrested cells with cosmid probes from a chromosome 19p contig available through the Lawrence Livermore National Laboratory (Livermore, CA). Analyses with a total of 16 cosmid clones resulted in the identification of cosmid R31546 that was split by the translocation (Fig. 1Go, b and c). A restriction map was constructed for der(15) and der(19) with the use of Southern blot analysis and the DNA sequences of cosmids R29783, R33209, and R31546, confirming the FISH mapping findings. The breakpoint was found to lie about 50 kilobases (kb) upstream of Notch3 and within the 3` untranslated region of a putative gene, Hunk1, on 19p. Since we were unable to demonstrate detectable expression of Hunk1 in HCC2429 cells or in other lung cancers in our panel and B-lymphoblastoid cell lines, Hunk1 is unlikely to be functionally activated by this translocation (data not shown).

In contrast, Notch3 messenger RNA was highly expressed in the HCC2429 cell line (Fig. 1Go, d; lane 2). Expression of Notch3 was detected in frozen tumor tissue from the index patient with the use of reverse transcription–polymerase chain reaction Southern blot analysis (data not shown). We further examined the expression of Notch3 in a total of 44 lung cancer cell lines (including HCC2429) representing all of the major lung cancer subtypes (Table 1Go). Seven of these cell lines were found to express Notch3 (Fig. 1Go, d). All of the Notch3-expressing lines had non-small-cell lung cancer or mesothelioma histology. Karyotype data were available on 29 of these 44 cell lines. All of the Notch-expressing cell lines for which karyotype data were available (six of seven) had karyotypic abnormalities involving chromosome 19 (P = .002, Fisher's exact test). The cell lines expressing Notch3 showed translocation of chromosome 19 to several other chromosome partners, including chromosomes 12q, 14q, 17q, 4q, and 6q. The precise location of the breakpoints on these chromosomes is currently unclear. One of these cell lines, NCI-H647, was studied by FISH and was found to have a 19p translocation within 150 kb 5` of the Notch3 start site, between cosmids R31546 and R29267 (data not shown). The Notch3-expressing cell lines were derived from patients with demographics more typical of those of patients with lung cancer than of our index case subject, including smokers and older patients. A multiple-tissue, northern blot analysis showed ubiquitous low-level expression of Notch3 in various tissues, including lung, brain, heart, aorta, kidney, thymus, and placenta, but a normal lung bronchial epithelial cell line showed no expression of Notch3 (data not shown). Consistent with this observation, Joutel et al. (9) recently demonstrated that Notch3 expression is limited to the vascular smooth muscle cells in all of these tissues, and they found no expression in lung epithelial cells with Notch3-specific antibodies. While the physiologic ligand for Notch3 is unknown, Delta-like, a known ligand, for Notch1, is expressed in the normal adult lung, raising the possibility of an autocrine loop (10).


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Table 1. Association of tumor cell line histology with 19p abnormalities and Notch3 overexpression
 
We have shown that Notch3 overexpression is associated with a translocation involving 19p, and overexpression is frequent in non-small-cell lung cancer. These data, together with knowledge of the involvement of other Notch family members in differentiation and neoplasia, make Notch3 a very strong candidate as the target of this translocation. Unlike Notch1 in leukemia, where t(7;9) rearrangement results in the overexpression of only the truncated intracellular domain, in all of the tumors studied here Notch3 appeared to be expressed as a full-length message. It is possible that a point mutation or abnormal processing could have resulted in an abnormal protein with a normal-sized message. Alternatively, the consequences of Notch activation may be different in cells of epithelial origin, or the mechanism of action of Notch3 per se may be different from that of other Notch family members. In addition to Notch's effects on differentiation, recent studies (1115) have linked the protein to regulation of apoptosis and the cell cycle.

In summary, to our knowledge, this is the first report that a specific chromosome translocation occurs in a more common epithelial cancer, such as lung carcinoma. Furthermore, the identified translocation activates a gene not previously implicated in lung cancer. The study of other chromosome translocations in epithelial tumors may identify additional novel transforming pathways and potential therapeutic targets that would be missed by studies of gene deletion or amplification.

NOTES

Note added in proof: Of the non-small-cell lung cancers that we tested, all of the Notch3-expressing tumors were adenocarcinomas except for one mesothelioma (H513). The American Type Culture Collection (Manassas, VA) stock of H513 is genetically identical to H125, an adenocarcinoma at all tested polymorphic loci. This finding strengthens the observed association of Notch3 with adenocarcinoma. We have additional preliminary data showing that Notch3 was expressed in the majority of a panel of 64 lung adenocarcinomas by immunohistochemistry of a tissue array.

Supported by Public Health Service grants 1P30CA68485 and P50CA70907 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (to the Vanderbilt-Ingram Cancer Center).

We thank Ms. Linda Ashworth and the Lawrence Livermore National Laboratory for providing the cosmid clones necessary for our cloning projects as well as their technical help. We also thank Dr. V. G. Dev and Edward Gonzales for their technical assistance in fluorescence in situ hybridization.

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Manuscript received January 3, 2000; revised May 22, 2000; accepted June 6, 2000.


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