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(Chest. 2005;128:2999-3003.)
© 2005 American College of Chest Physicians

Identification of Tumor Suppressor Loci on the Long Arm of Chromosome 5 in Pulmonary Large Cell Neuroendocrine Carcinoma*

Ju Hye Shin; Shin Myung Kang, MD; Young Sam Kim, MD, PhD; Dong Hwan Shin, MD, PhD; Joon Chang, MD, PhD; Sung Kyu Kim, MD, PhD and Se Kyu Kim, MD, PhD

* From the Cancer Metastasis Research Center (Drs. JH Shin and Se Kyu Kim), and the Department of Internal Medicine (Drs. Kang, YS Kim, Chang, and Sung Kyu Kim) and Pathology (Dr. DH Shin), Yonsei University College of Medicine, Seoul, Korea.

Correspondence to: Se Kyu Kim, MD, PhD, Department of Internal Medicine, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Gu, Seoul, 120–752, Korea; e-mail: sekyukim{at}yumc.yonsei.ac.kr


   Abstract
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 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
Background: A recent cytogenetic analysis of non-small cell lung cancer revealed hot-spot regions for deletion on the long arm of chromosome 5 and suggested the existence of putative tumor suppressor genes in that region. However, similar studies on genetic alterations in large cell neuroendocrine carcinoma (LCNEC) have been very limited. To our knowledge, this is the first report to screen for the loss of heterozygosity (LOH) and to examine the location of putative tumor suppressor genes on chromosome 5q in LCNEC.

Objectives: To identify tumor suppressor loci on chromosome 5q in LCNEC by microsatellite analysis.

Patients and methods: Microsatellite instability and LOH in tumor and normal tissue samples from 13 patients with LCNEC, who had undergone surgical resections, were analyzed by polymerase chain reaction using a panel of 19 microsatellite DNA markers spanning chromosome 5q.

Results: LOH was found in all of the 13 tumors (100%) in at least one informative marker tested. The following four common minimally deleted regions were noticed on chromosome 5q: 5q14.3-q21.1; 5q22.2-q23.1; 5q23.3-q33.2; and 5q35.1-q35.2. Three of 13 individual tumors (23.1%) exhibited shifted bands for at least one of the tested microsatellite markers. Shifted bands occurred in 6 of 224 loci (2.7%) tested.

Conclusion: These data suggest the presence of at least four tumor suppressor loci on chromosome 5q in LCNEC, and further investigations into cloning candidate tumor suppressor genes are warranted.

Key Words: chromosome 5q • large cell neuroendocrine carcinoma • loss of heterozygosity • tumor suppressor


   Introduction
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 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
Lung cancer is the leading cause of cancer death in the United States. Large cell neuroendocrine carcinoma (LCNEC) is one of the variants of large cell carcinoma and is one of the primary lung cancers portending poor survival for patients.123 It is well-known that the accumulation of genetic and epigenetic changes, indicating the inactivation of tumor suppressor genes and the activation of oncogenes, occurs during the process of carcinogenesis and subsequent progression of lung cancer.456 Thus, it is valuable to identify the putative tumor suppressor loci that may play roles in lung carcinogenesis, especially in high-grade malignant tumors.

The long arm of chromosome 5 (5q) represents a common target for genetic alterations in non-small cell lung cancer (NSCLC).7 The adenomatous polyposis coli (APC) and mutated in colorectal cancer (MCC) genes are well-known tumor suppressor genes on chromosome 5q, and alterations in these genes are common events in NSCLC.8910 A cytogenetic analysis of NSCLCs showed additional hot-spot deletion regions on chromosome 5q and suggested the existence of putative tumor suppressor genes.111213 However, the studies141516 on genetic alterations in LCNECs have been very limited, and only a few microsatellite markers that show frequent loss of heterozygosity (LOH) in small cell lung cancers (SCLCs) were used in those studies. To examine the potential presence of other tumor suppressor genes on chromosome 5q, we tested 13 primary LCNECs by intensive microsatellite analysis. To our knowledge, this is the first report to screen for LOH and to examine the location of putative tumor suppressor genes on chromosome 5q using 19 microsatellite markers covering the panel of LCNECs.


   Methods and Materials
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 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Primary Tumors and Control Tissues
Paraffin-embedded tissue blocks containing primary LCNECs were generated after the surgical resection of 13 primary tumors at The Severance Hospital, Yonsei University College of Medicine, Seoul, Korea (Table 1 ). DNA was extracted from primary tumor tissues and corresponding control tissues. The control tissues were obtained from normal adjacent tissue. Paraffin-embedded tissue blocks were sectioned using a microtome. A 4-µm section from each block was stained with hematoxylin-eosin and was reviewed by pathologists to confirm the diagnosis, locate tumor areas, and select tumor-free control samples. Four or five additional 10-µm serial sections were mounted on slides and stained with hematoxylin-eosin. Tumors were dissected with a scalpel under a stereomicroscope to ensure the accuracy of the dissection. We used only the tumor blocks in which tumor cells could be dissected with < 30% normal cell contamination. Normal control tissues were dissected using the same approach.


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  		Table 1.. Characteristics of 13 Patients With LCNEC

 
DNA Extraction and Microsatellite Analysis
Dissected tissues were digested in 200 µL of 50 mmol/L Tris-HCl (pH 8.0) containing 1% sodium dodecyl sulfate-proteinase K and incubated at 42°C for 12 to 24 h. The digested products were purified by treatment with phenol-chloroform. DNA was precipitated using the ethanol precipitation method in the presence of glycogen. For microsatellite analysis, 19 microsatellite markers on chromosome 5q (including D5S118, D5S107, D5S644, D5S409, D5S346, D5S421, D5S404, FBN 2, IRF-1, IL-9, D5S414, D5S178, D5S210, D5S209, D5S820, D5S400, D5S625, D5S429, and D5S498) were obtained (Research Genetics; Huntsville, AL). One of the primers for each marker was end-labeled with [{gamma}-32P] ATP (3,000Ci/mmol; Buckinghamshire, UK) and T4 DNA polynucleotide kinase (New England Biolabs; Beverly, MA). Polymerase chain reactions (PCRs) were carried out in a 12.5-µL volume of a solution containing 20 ng of genomic DNA, 1% dimethylsulfoxide, 200 µmol/L deoxynucleoside triphosphate, 1.5 mmol/L MgCl2, 0.4 µmol/L PCR primers, including 0.1 µmol/L {gamma}-32P-labeled primer, and 0.5 U of Taq DNA polymerase (GIBCO-BRL; Gaithersburg, MD). DNA was amplified for 35 cycles at 95°C for 30 s, 52 to 60°C for 60 s, and 70°C for 60 s in a temperature cycler (Hybaid; Omnigene; Woodbridge, NJ) in 500-µL plastic tubes, followed by a 5-min extension at 70°C. The PCR products were separated on a 6% polyacrylamide-urea-formamide gel, which was then autoradiographed. LOH was defined as a > 50% reduction of the intensity by visual inspection in either of the two alleles compared with those in normal control panels. Shifted bands were determined by the appearance of clear novel alleles that were not observed in normal tissue control panels.


   Results
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 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
LOH frequencies at individual markers tested are summarized in Table 2 . LOH was found in all of the 13 tumors (100%) in at least one of the informative marker tested. Four common minimally deleted regions were located between the markers D5S107 and D5S409 at 5q14.3-q21.1, D5S421and D5S404 at 5q22.2-q23.1, IRF-1 and D5S209 at 5q23.3-q33.2, and D5S400 and D5S498 at 5q35.1-q35.2 (Fig 1 ). Three of 13 individual tumors (23.1%) exhibited shifted bands for at least one of the tested microsatellite markers. Shifted bands occurred in 6 of 224 loci (2.7%) tested. The examples of LOH at different regions are shown in Figure 2 .


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  		Table 2.. Frequencies of LOH in Chromosome 5q of LCNEC

 


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  		Figure 1.. Schematic representation of the deletion map of chromosome 5q in LCNEC. The minimal deleted regions are indicated on the right side of the figure.

 


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  		Figure 2.. Examples of deletions observed in LCNEC. Left, A: order of the microsatellite markers used in this study. Middle, B: LOH at markers D5S644, D5S421, D5S178, and D5S429, and retention at markers D5S346 and D5S820 in tumor LCNEC7. Right, C: LOH at markers D5S107, D5S404, and IL-9, and retention at markers D5S346, FBN2, and D5S820 in tumor LCNEC8. N = normal tissues; T = tumor tissues.

 

   Discussion
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 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
According to the two-hit hypothesis of Knudson,17 inducing a loss of function in tumor suppressor genes requires that both alleles should be inactivated. One allele is inactivated by allelic loss, whereas the other allele is inactivated by point mutation, aberrant methylation, or other changes that target the individual tumor suppressor gene. Thus, consistent LOH for genetic markers at a given locus in many tumors is strong evidence for the presence of one or more tumor suppressor genes in that region.57

In 1999, LCNEC was categorized as one of the variants of large cell carcinoma based on the histologic criteria proposed by Travis et al.1 LCNECs represent only 1 to 2% of malignant pulmonary neoplasms18 but share various clinicopathologic and biological features with SCLC, such as high-grade malignant neuroendocrine tumors and a poor patient prognosis. Thus, clinical studies231819 on LCNEC have been compared with those on SCLC, and studies1416 on genetic alterations associated with LCNEC have been limited to the few markers that have been frequently used in SCLC studies.

Chromosome 5q represents a common target for genetic alterations in NSCLC.7 In addition to SCLC and NSCLC, LOH on chromosome 5q has been identified in ovarian cancer, stomach cancer, esophageal cancer, myelodysplastic syndrome, and testicular germ cell tumors.2021222324 Chromosome 5q showed frequent deletions of the chromosome arm in LCNEC.15 APC/MCC genes are well-known tumor suppressor genes on chromosome 5q. Some cytogenetic studies111213 reported other hot-spot regions for deletion in NSCLC, which were 5q14, 5q31.1, and 5q34. These regions suggest the existence of putative tumor suppressor genes on chromosome 5q.

We analyzed the panel of LCNECs using 19 microsatellite markers on chromosome 5q and identified four distinct, minimally deleted regions on chromosome 5q in this tumor type. One hundred percent of the tumors (13 of 13 tumors) lost at least one of the informative markers tested. All of the 13 tumors showed LOH in more than two distinct regions on chromosome 5q.

The first commonly deleted region is located between markers D5S107 and D5S409 at 5q14.3-q21.1, a distance of 19.1 centimorgan (cM). 5q14 is one of the hot-spot regions in chromosome 5. In this region, a high frequency of LOH is observed in ovarian cancer, testicular germ cell tumors, and gastric cancer.202325 Additionally, some studies1213 of NSCLC employing the D5S644 microsatellite marker have shown a high frequency of LOH in this region. In our study, 50% of LCNECs tested at D5S644 displayed LOH, and a very high frequency (91.7%) of LOH was observed in the region of 5q14.3-q21.1. The second commonly deleted region identified in this study is located between markers D5S421 and D5S404 at 5q22.2-q23.1, a distance of 4.0 cM. The APC gene and MCC gene are two well-known tumor suppressor genes on chromosome 5q. In addition to colon cancer, a high frequency of LOH at this region has been reported in NSCLC and SCLC.8262728 Onuki and colleagues16 reported that 46% of LCNECs showed LOH at this region. A high frequency of LOH at the second commonly deleted region located between markers D5S421 and D5S404, which is about 0.7 cM telomeric to the APC gene and 0.3 cM telomeric to the MCC gene, may suggest the association between LOH and the APC/MCC genes. However, the frequency of LOH at marker D5S346, which is 0.03 cM telomeric to APC/MCC genes, was low (16.7%) in our study. This implies that another tumor suppressor gene, different from the APC/MCC genes, may be present in LCNECs and that further evaluations are required.

The third commonly deleted region is located between IRF-1 and D5S209 at 5q23.3-q33.2, a distance of 20.7 cM. In this region, it was reported that 30% of LCNECs showed LOH.6 This region encompasses the gene encoding the interferon regulatory factor (IRF) 1 locus at 5q31.1. IRF-1 is a transcriptional activator that shows tumor suppressor activity.29 In human cancers, it has been observed that functional IRF-1 messenger RNA expression was not detectable in acute leukemia or in myelodysplastic syndrome.30 In addition, a functionally inactivating point mutation in the IRF-1 gene was identified in gastric cancer.31 A high frequency of LOH on 5q31.1 was also reported in NSCLC.13 Accordingly, these data, together with the fact that 100% of the LCNECs lost a region close to the IRF-1 gene in our study, suggest that the IRF-1 gene is an excellent candidate tumor suppressor gene in LCNEC.

The fourth commonly deleted region identified in this study is between markers D5S400 and D5S498 at 5q35.1-q35.2, a distance of 5.4 cM. More than 84% of LCNECs in our tumor panel lost this region, suggesting the presence of a tumor suppressor gene. The frequent losses of similar regions have also been reported in SCLC and NSCLC.1113 However, no tumor suppressor genes have currently been located around this region. Three tumors (LCNEC10, LCNEC11, and LCNEC12) exhibited a clear loss at marker D5S429, while the allele was retained at markers D5S625 and D5S498 (Fig 1). This indicated that a candidate tumor suppressor gene may be located at or more centromeric to the marker D5S498.

The data from this study suggest the presence of at least four tumor suppressor loci on chromosome 5q, and they may play important roles in the tumor development and cancer progression processes in LCNEC. Further intensive studies to clone the candidate tumor suppressor genes are warranted.


   Footnotes
 
Abbreviations: APC = adenomatous polyposis coli; cM = centimorgan; IRF = interferon regulatory factor; LCNEC = large cell neuroendocrine carcinoma; LOH = loss of heterozygosity; MCC = mutated in colorectal cancer; NSCLC = non-small cell lung cancer; PCR = polymerase chain reaction; SCLC = small cell lung cancer

Dr. Ss Kyu Kim had full access to all of the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis.

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Cancer Metastasis Research Center (CMRC) at Yonsei University.

Received for publication March 20, 2005. Accepted for publication April 27, 2005.


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