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Cancer Research 67, 11601, December 15, 2007. doi: 10.1158/0008-5472.CAN-07-3243
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Cancer-Testis Antigen Lymphocyte Antigen 6 Complex Locus K Is a Serologic Biomarker and a Therapeutic Target for Lung and Esophageal Carcinomas

Nobuhisa Ishikawa1,2, Atsushi Takano1, Wataru Yasui3, Kouki Inai4, Hitoshi Nishimura5, Hiroyuki Ito6, Yohei Miyagi7, Haruhiko Nakayama6, Masahiro Fujita8, Masao Hosokawa8, Eiju Tsuchiya7, Nobuoki Kohno2, Yusuke Nakamura1 and Yataro Daigo1

1 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Departments of 2 Molecular and Internal Medicine, 3 Molecular Pathology, and 4 Pathology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 5 Department of Thoracic Surgery, Saitama Cancer Center, Saitama, Japan; 6 Division of Thoracic Surgery and 7 Molecular Pathology and Genetics Division, Kanagawa Cancer Center, Kanagawa, Japan; and 8 Keiyukai Sapporo Hospital, Sapporo, Japan

Requests for reprints: Yataro Daigo, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5457; Fax: 81-3-5449-5406; E-mail: ydaigo{at}ims.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene expression profile analyses of non–small cell lung carcinomas (NSCLC) and esophageal squamous cell carcinomas (ESCC) revealed that lymphocyte antigen 6 complex locus K (LY6K) was specifically expressed in testis and transactivated in a majority of NSCLCs and ESCCs. Immunohistochemical staining using 406 NSCLC and 265 ESCC specimens confirmed that LY6K overexpression was associated with poor prognosis for patients with NSCLC (P = 0.0003), as well as ESCC (P = 0.0278), and multivariate analysis confirmed its independent prognostic value for NSCLC (P = 0.0035). We established an ELISA to measure serum LY6K and found that the proportion of the serum LY6K-positive cases was 38 of 112 (33.9%) NSCLC and 26 of 81 (32.1%) ESCC, whereas only 3 of 74 (4.1%) healthy volunteers were falsely diagnosed. In most cases, there was no correlation between serum LY6K and conventional tumor markers of carcinoembryonic antigen (CEA) and cytokeratin 19-fragment (CYFRA 21-1) values. A combined ELISA for both LY6K and CEA classified 64.7% of lung adenocarcinoma patients as positive, and the use of both LY6K and CYFRA 21-1 increased sensitivity in the detection of lung squamous cell carcinomas and ESCCs up to 70.4% and 52.5%, respectively, whereas the false positive rate was 6.8% to 9.5%. In addition, knocked down of LY6K expression with small interfering RNAs resulted in growth suppression of the lung and esophageal cancer cells. Our data imply that a cancer-testis antigen, LY6K, should be useful as a new type of tumor biomarker and probably as a target for the development of new molecular therapies for cancer treatment. [Cancer Res 2007;67(24):11536–46]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aerodigestive tract cancer (including carcinomas of lung, esophagus, oral cavity, pharynx, and larynx) accounts for one third of all cancer deaths in the United States and is the most common cancer in some areas of the world (1). Lung cancer is one of the most common malignant tumors in the world, and non–small cell lung cancer (NSCLC) accounts for nearly 80% of those cases (2). Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies of the digestive tract, and at the time of diagnosis, most of the patients are at advanced stages (3). In spite of the use of modern surgical techniques combined with various adjuvant treatment modalities, such as radiotherapy and chemotherapy, the overall 5-year survival rate of ESCC patients still remains at 40% to 60% (4) and that of lung cancer patients is only 15% (5, 6), although a reduced quality of life remains in those who do survive. Several tumor markers, such as progastrin-releasing peptide, neuron-specific enolase, cytokeratin 19-fragment (CYFRA 21-1), squamous cell carcinoma antigen (SCC), and carcinoembryonic antigen (CEA) are shown to be elevated in serum of lung cancer patients (7), whereas SCC, CEA, and CYFRA 21-1 are elevated in the serum of advanced ESCC patients (8). However, their sensitivity remains at 20% to 50% and no tumor marker has been sufficiently useful for detection of lung cancer and ESCC at potentially curative stage, and a limited number of practical prognostic biomarker is presently available for selection of treatment modalities for individual patients. Therefore, new diagnostic tools and therapeutic strategies, such as development of molecular targeted agents, antibody therapy, and cancer vaccines, are urgently required (6).

Cancer-testis antigens are defined to be proteins that are highly expressed in cancer cells but not in normal cells, except for cells in reproductive tissues, such as testis, ovary, and placenta (9, 10). Because the cells from these tissues do not express MHC class I molecules, cancer-testis antigens are considered to be a promising target for immunotherapy, such as cancer vaccines, and also a good biomarker for diagnosis of cancer and monitoring of relapse.

Systematic analysis of expression levels of thousands of genes using a cDNA microarray technology is an effective approach for identifying molecules involved in pathways of carcinogenesis or those associated with efficacy to anticancer therapy (11–16); some of such genes or their gene products may be good target molecules for development of novel therapies and/or cancer biomarkers. To identify such molecules, particularly for cancer-testis antigens, we had performed genome-wide expression profile analysis of 101 lung cancer and 19 ESCC patients, coupled with enrichment of tumor cells by laser capture microdissection (12–16) and then compared with the expression profile data of 31 normal human tissues (27 adult and 4 fetal organs; refs. 17, 18). To verify the biomedical and clinicopathologic significance of the respective gene products, we have established a screening system by a combination of the tumor-tissue microarray analysis of clinical lung and esophageal cancer materials and RNA interference technique (19–33). This systematic approach revealed that lymphocyte antigen 6 complex locus K (LY6K; alias HSJ001348, cDNA for differentially expressed CO16 gene), a member of LY6 family, is likely to be a novel cancer-testis antigen that was overexpressed commonly in primary NSCLCs and ESCCs and was essential for growth/survival of cancer cells.

LY6K was initially identified as an unannotated transcript by several groups (accession no. AJ001348; AB105187), and recent analysis by Bioinformatics classified it as a member belonging to the LY6 family that shows a high homology to the low–molecular weight GPI-anchored molecule (34). A previous study showed the elevated expression of LY6K mRNA in human head and neck squamous cell carcinomas and breast cancers (34, 35); however, no report has clarified the significance of activation of LY6K in human cancer progression and its potential as a therapeutic target, as well as serologic/prognostic biomarker.

Here, we report evidence that LY6K, a member of cancer-testis antigen, plays a significant role in pulmonary and esophageal carcinogenesis, and suggest that LY6K could be a new type of diagnostic and prognostic biomarker that could detect certain proportion of cancer patients that may not be diagnosed by conventional tumor makers in the clinic.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tissue samples. The 18 human NSCLC cell lines, 14 esophageal carcinoma cell lines, and human small airway epithelial cells were used as described elsewhere (19, 30). Primary NSCLC and ESCC samples had been obtained earlier with informed consent (refs. 15, 16, 25, 30; see Supplementary Materials and Methods). A total of 406 formalin-fixed samples of primary NSCLCs and adjacent normal lung tissues had been obtained earlier along with clinicopathologic data from patients undergoing surgery at Saitama Cancer Center. These NSCLC patients received resection of their primary cancers without preoperative chemotherapy and/or radiotherapy, and among them, only patients with positive lymph-node metastasis were treated with cisplatin-based adjuvant chemotherapies after their surgery. A total of 265 formalin-fixed primary ESCCs and adjacent normal esophageal tissue samples had also been obtained from patients undergoing curative surgery at Keiyukai Sapporo Hospital. These ESCC patients received resection of their primary cancers without preoperative chemotherapy and/or radiotherapy. NSCLC specimen and five tissues (heart, liver, lung, kidney, and testis) from postmortem materials (two individuals with SCC) were also obtained from Hiroshima University. The pathologic stage was determined according to the classification of the Union Internationale Contre le Cancer (36). This study and the use of all clinical materials mentioned were approved by individual institutional ethical committees.

Serum samples. Serum samples were obtained with informed consent from 74 healthy individuals as controls and from 65 nonneoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) enrolled as a part of the Japanese Project for Personalized Medicine (BioBank Japan) or admitted to Hiroshima University Hospital. All of these COPD patients were current and/or former smokers. [The mean (±1 SD) of pack-year index was 55.6 ± 50.1 SD; pack-year index was defined as the number of cigarette packs (20 cigarettes per pack) consumed a day multiplied by years]. Serum samples were also obtained with informed consent from 112 NSCLC patients admitted to Hiroshima University Hospital, as well as Kanagawa Cancer Center Hospital, and 81 esophageal cancer patients, who were admitted to Keiyukai Sapporo Hospital or who were registered in the BioBank Japan. These 112 NSCLC cases included 85 adenocarcinomas and 27 SCCs (see Supplementary Materials and Methods). Samples were selected for the study on the basis of the following criteria: (a) patients were newly diagnosed and previously untreated and (n) their tumors were pathologically diagnosed as lung or esophageal cancers (stages I-IV). Serum was obtained at the time of diagnosis and stored at –150°C.

Semiquantitative reverse transcription–PCR and Northern blot analyses. We performed semiquantitative reverse transcription–PCR (RT-PCR) and Northern blot analyses as described in Supplementary Materials and Methods.

Preparation of anti-LY6K polyclonal antibody. Two types of rabbit antibodies termed TM38 and MB44 specific for LY6K were raised by immunizing rabbits with 6-histidine fused human LY6K protein (codons 23-109 and 71-204, respectively) and purified with standard protocols using affinity columns (Affi-gel 10; Bio-Rad Laboratories) conjugated with the 6-histidine fused protein. On Western blots, we confirmed that the antibodies were specific for LY6K, using lysates from NSCLC tissues and cell lines, as well as normal lung tissues.

Western blot analysis. We used an enhanced chemiluminescence Western blotting analysis system (GE Healthcare Biosciences). SDS-PAGE was performed in 7.5% polyacrylamide gels. PAGE-separated proteins were electroblotted onto nitrocellulose membranes (GE Healthcare Biosciences) and incubated with a rabbit polyclonal antihuman LY6K antibody. A goat antirabbit IgG–horseradish peroxidase (HRP) antibody (GE Healthcare Biosciences) was served as the secondary antibodies for these experiments.

Immunohistochemistry and tissue microarray. Tumor-tissue microarrays were constructed using 406 formalin-fixed primary NSCLCs and 265 ESCCs, as published previously (see Supplementary Materials and Methods; refs. 37–39).

To investigate the status of the LY6K protein in clinical lung-cancer samples that had been embedded in paraffin blocks, we stained the sections in the following manner. Briefly, a rabbit polyclonal antihuman LY6K antibody (TM38) was added after blocking of endogenous peroxidase and proteins. The sections were incubated with HRP-labeled antirabbit IgG as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.

Three independent investigators assessed LY6K positivity semiquantitatively without prior knowledge of clinicopathologic data. The intensity of LY6K staining was evaluated using the following criteria: strong positive (2+), dark brown staining in >50% of tumor cells completely obscuring cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cells; absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted only as strongly positive if reviewers independently defined them as such.

Statistical analysis. We used contingency tables to analyze the relationship of LY6K expression levels and clinicopathologic variables of NSCLC patients. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for LY6K expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were performed with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality (see Supplementary Materials and Methods).

ELISA. Serum levels of LY6K were measured by sandwich-type ELISA, which had been originally constructed. In brief, for detection of soluble LY6K in serum, 96-well flexible microtiter plates (Nalge Nunc International) were coated with 2 ng/mL of capturing polyclonal antibody to LY6K (TM38) overnight. Wells were blocked with 200 µL PBS (pH 7.4) containing 1% bovine serum albumin (BSA), 5% sucrose, and 0.05% NaN3 for 2 h and then incubated for 2 h with 3-fold diluted serum samples in PBS (pH 7.4) containing 1% BSA. After washing with PBS (pH 7.4) containing 0.05% Tween 20, the wells were incubated for 2 h with 200 ng/mL of biotin-conjugated polyclonal anti-LY6K antibody (MB44), followed by reaction with avidin-conjugated peroxidase (P347; Dako Cytomation) for 30 min using a substrate reagent (R&D Systems). To prepare biotinylating rabbit polyclonal antibodies to LY6K (MB44), we used Biotin Labeling Kit-NH2 (LK03) according to the supplier's protocol (Dojindo Laboratories). Levels of CEA in serum were measured by ELISA with a commercially available enzyme test kit (Hope Laboratories, Belmont, CA), according to the supplier's recommendations. Levels of CYFRA 21-1 in serum were measured by ELISA with a commercially available kit (DRG; see Supplementary Materials and Methods).

RNA interference assay. We had previously established a vector-based RNA interference system, psiH1BX3.0, to direct the synthesis of small interfering RNAs (siRNA) in mammalian cells (see Supplementary Materials and Methods; refs. 19, 21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LY6K expression in lung and esophageal tumors, cell lines, and normal tissues. To identify novel molecules, such as cancer-testis antigens to serve as diagnostic biomarkers and/or targets for development of therapeutic agents for lung and esophageal cancers, we had applied cDNA microarray analysis to search for genes that were highly transactivated in a large proportion of these tumors, but scarcely expressed in normal tissues. Among 27,648 genes screened, we identified the LY6K transcript to show >3-fold expression in ~80% of NSCLCs and ESCCs compared with normal lung or esophageal tissue (control). Moreover, LY6K showed testis-specific expression in normal tissues. Therefore, we determined to select LY6K as a good candidate for further analyses. We confirmed its transactivation by semiquantitative RT-PCR experiments in 9 of 10 additional NSCLC tissues, in 10 of 18 lung-cancer cell lines, in 8 of 8 ESCC tissues, and in 12 of 14 esophageal cancer cell lines (Fig. 1A and B ).


Figure 1
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  		Figure 1. Expression and subcellular localization of LY6K in NSCLCs and ESCCs. A, expression of LY6K in 10 clinical NSCLC samples (top) and 18 lung cancer cell lines (bottom) examined by semiquantitative RT-PCR analysis. B, expression of LY6K in eight clinical ESCC samples (top) and 14 esophageal cancer cell lines (bottom), as detected by semiquantitative RT-PCR analysis. C, expression of LY6K protein in four representative pairs of NSCLC samples (left) and four lung cancer cell lines (right) examined by Western blot analysis. D, subcellular localization of endogenous LY6K protein in lung cancer cells (left). LY6K is stained at the cytoplasm of the cell with granular appearance in LC319 and NCI-H1373 cells, but not in NCI-H226 and A427 cells (right). Measurement of secreted LY6K levels with ELISA in culture medium of LY6K-expressing LC319 and NCI-H1373 cells and nonexpressing NCI-H226 and A427 cells.

 
We subsequently generated rabbit polyclonal antibodies specific for human LY6K and confirmed by Western blot analysis an expression of LY6K protein in NSCLC samples in four representative pairs of NSCLC tissues and in four lung-cancer cell lines (two LY6K-positive and two LY6K-negative cell lines; Fig. 1C). We performed immunofluorescence analysis to examine the subcellular localization of endogenous LY6K in the four lung-cancer cell lines (LC319, NCI-H1373, NCI-H226, and A427) and found that LY6K was mainly located at cytoplasm of tumor cells with granular appearance (Fig. 1D, left). Because LY6K encodes GPI-anchored protein and some of GPI-anchored proteins were known to be secreted into extracellular space (40), we applied ELISA to examine its presence in the culture media of the lung-cancer cell lines. The amounts of detectable LY6K in the culture media was concordant to the expression levels of LY6K detected with semiquantitative RT-PCR and Western blot analyses (Fig. 1D, right).

Northern blot analysis using a LY6K cDNA fragment as the probe identified a transcript of ~1.8 kb that was highly and exclusively expressed in testis among 23 normal human tissues examined (Fig. 2A ). We subsequently examined expression of LY6K protein in five normal tissues (heart, liver, lung, kidney, and testis), as well as lung cancers using anti-LY6K antibody, and found that it was hardly detectable in the former four tissues while positive LY6K staining appeared in testis and lung tumor tissues (Fig. 2B).


Figure 2
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  		Figure 2. Expression of LY6K in normal tissues, as well as NSCLC and ESCC tissues, and association of LY6K overexpression with poor clinical outcomes for NSCLC and ESCC patients. A, Northern blot analysis of the LY6K transcript in 23 normal adult human tissues. B, immunohistochemical evaluation of LY6K protein in representative normal tissues: adult heart, liver, lung, kidney, and testis, as well as lung SCC tissues. C, immunohistochemical evaluation of LY6K expression on tumor tissue microarrays (top, x100; bottom, x200). Examples are shown of strong, weak, and absent LY6K expressions in cancer tissues and of no expression in normal tissues. Top, lung SCC and normal lung; bottom, ESCC and normal esophagus. D, Kaplan-Meier analysis of survival of patients with NSCLC (left, P = 0.0003 by the log-rank test) and ESCC (right, P = 0.0278 by the log-rank test) according to the expression levels of LY6K.

 
Association of LY6K overexpression with poor clinical outcomes for NSCLC and ESCC patients. To verify the biological and clinicopathologic significance of LY6K, we examined the expression of LY6K protein by means of tissue microarrays consisting of 406 NSCLC and 265 ESCC cases that underwent surgical resection. LY6K staining was observed mainly in the cell membrane and cytoplasm of tumor cells, but was hardly detectable in surrounding normal tissues (Fig. 2C, top and bottom). We classified a pattern of LY6K expression on the tissue array ranging from absent/weak (scored as 0-1+) to strong (2+). Positive staining was observed in 219 of 254 (86.2%) lung adenocarcinoma cases, 103 of 112 (92.0%) lung SCCs, 24 of 28 (85.7%) lung large cell carcinomas, and 12 of 12 (100%) lung adenosquamous carcinomas, whereas no staining was observed in any of the normal portions of the same tissues. Of the 406 NSCLC cases examined, LY6K was strongly stained in 139 cases (34.2%; score 2+), weakly stained in 219 cases (54.0%; score 1+), and not stained in 48 cases (11.8%; score 0; details were shown in Table 1A ). NSCLC patients whose tumors showed strong LY6K expression revealed shorter tumor-specific survival compared with those with absent/weak LY6K expression (P = 0.0003 by log-rank test; Fig. 2D, left). We also applied univariate analysis to evaluate associations between patient prognosis and other factors, including age (<65 versus ≥65), gender (female versus male), histologic type (adenocarcinoma versus nonadenocarcinoma), pT classification (T1, T2 versus T3, T4), pN classification (N0 versus N1, N2), and LY6K status (0, 1+ versus 2+). Among those variables, LY6K status (P = 0.0004), elderly (P = 0.0093), male (P = 0.0050), nonadenocarcinoma histologic classification (P = 0.0108), advanced pT stage (P < 0.0001), and advanced pN stage (P < 0.0001) were significantly associated with poor prognosis (Table 1B). In multivariate analysis of the prognostic factors, strong LY6K expression, elderly, higher pT stage, and higher pN stage were indicated to be independent prognostic factors (P = 0.0035, < 0.0001, < 0.0001, and < 0.0001, respectively; Table 1B).


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  		Table 1.

 
Positive staining was observed in 252 of 265 (95.1%) esophageal cancers, whereas no staining was observed in any of the normal portions of the same tissues. LY6K was strongly stained in 175 cases (66.0%; score 2+), weakly stained in 77 cases (29.1%; score 1+), and not stained in 13 cases (4.9%; score 0; details were shown in Table 2A ). The median survival time of ESCC patients was significantly shorter in accordance with the higher expression levels of LY6K (P = 0.0278 by log-rank test; Fig. 2D, right). We also applied univariate analysis to evaluate associations between ESCC patient prognosis and several factors including age (<65 versus ≥65), gender (female versus male), pT stage (tumor depth; T1, T2 versus T3, T4), pN stage (node status; N0 versus N1), and LY6K status (score; 0, 1+ versus 2+). Among those variables, LY6K status (P = 0.0289), male (P = 0.0029), advanced pT stage (P < 0.0001), and advanced pN stage (P < 0.0001) were significantly associated with poor prognosis (Table 2B). In multivariate analysis, LY6K status did not reach the statistically significant level as an independent prognostic factor for surgically treated ESCC patients enrolled in this study (P = 0.2266), whereas pT and pN stages, as well as male gender, did, suggesting the relevance of LY6K expression to these clinicopathologic factors in esophageal cancer (P = 0.0149, 0.0047, and <0.0001, respectively; Table 2B).


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  		Table 2.

 
Serum levels of LY6K in patients with NSCLC or ESCC. Because the in vitro findings had suggested that LY6K could be secreted into extracellular space (Fig. 1D, right), we examined whether LY6K was secreted into serum from patients with NSCLC or ESCC to validate its potential as a novel serum biomarker. ELISA experiments detected LY6K in serologic samples from the great majority of the 193 patients with lung or esophageal cancer. The mean (±1 SD) of serum LY6K in 112 lung cancer patients was 331.3 ± 739.3 pg/mL and those in 81 ESCC patients were 209.3 ± 427.4 pg/mL. In contrast, the mean (±1 SD) serum levels of LY6K in 74 healthy individuals were 34.2 ± 65.3 pg/mL, and those in 65 patients with COPD, who were current and/or former smokers, were 54.4 ± 233.8 pg/mL. The levels of serum LY6K protein were significantly higher in lung or esophageal cancer patients than in healthy donors (between lung adenocarcinoma patients and healthy individuals, P < 0.0001; between lung SCCs and healthy individuals, P = 0.0145; between ESCCs and healthy individuals, P < 0.0001; Mann-Whitney U test), whereas the difference between healthy individuals and COPD patients was not significant (P = 0.5325; Fig. 3A ). According to histologic types of lung cancer, the mean (±1 SD) serum levels of LY6K were 324.1 ± 737.4 pg/mL in 85 adenocarcinoma patients and 354.1 ± 758.8 pg/mL in 27 SCC patients; the differences between the two histologic types were not significant. High levels of serum LY6K were detected even in patients with earlier-stage tumors (Fig. 3B). Using receiver-operating characteristic curves drawn with the data of these 193 lung or esophageal cancer patients and 74 healthy donors (data not shown), the cutoff level in this assay was set to provide optimal diagnostic accuracy and likelihood ratios (minimal false negative and false positive results) for LY6K, i.e., 157.0 pg/mL with a sensitivity of 33.2% (64 of 193) and a specificity of 4.1% (3 of 74). According to tumor histology, the proportions of the serum LY6K-positive cases were 31.8% for adenocarcinoma (27 of 85), 40.7% for SCC (11 of 27), and 32.1% for ESCC (26 of 81). The proportions of the serum LY6K-positive cases were 9.2% (6 of 65) for COPD. We then performed ELISA experiments using paired preoperative and postoperative (2 months after the surgery) serum samples from lung cancer and ESCC patients to monitor the levels of serum LY6K in the same patients. The concentration of serum LY6K was significantly reduced after surgical resection of primary tumors (Fig. 3C). We further compared the serum LY6K values with the expression levels of LY6K in primary tumors in the same set of 16 NSCLC cases whose serum had been collected before surgery (eight patients with LY6K-positive tumors and eight with LY6K-negative tumors). The levels of serum LY6K showed good correlation with the expression levels of LY6K in primary tumor (Fig. 3D). The results independently support the high specificity and the great potentiality of serum LY6K as a biomarker for detection of cancer at an early stage and for monitoring of the relapse of the disease.


Figure 3
Figure 3
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  		Figure 3. Serologic concentration of LY6K determined by ELISA in serum of patients with lung cancers or esophageal cancers and in healthy controls or nonneoplastic lung disease patients with COPD. A, distribution of LY6K in sera from patients with lung adenocarcinoma (ADC), lung SCC, and ESCC. Averaged serum levels are shown under the panel. Differences were significant between lung adenocarcinoma patients and healthy individuals (P < 0.0001, Mann-Whitney U test), between lung SCC patients and healthy individuals (P = 0.0145), and between ESCC patients and healthy individuals (P < 0.0001). B, distribution of LY6K in sera from patients at various clinical stages of lung cancers. C, serologicconcentration of LY6K before and aftersurgery (postoperative days at 2 mo)in patients with NSCLC and ESCC.Dotted line, cutoff level for LY6K (157.0pg/mL). D, serum LY6K levels (pg/mL) andthe expression levels of LY6K in primarytumor tissues in the same NSCLC patients.Score, intensity of LY6K staining thatwas evaluated using the criteria describedin Materials and Methods.

 
Comparison of LY6K with CEA and CYFRA 21-1 as tumor biomarkers. To evaluate the feasibility of using serum LY6K level as a tumor detection biomarker, we also measured by ELISA serum levels of two conventional tumor markers (CEA and CYFRA 21-1 for NSCLC patients) in the same set of available serum samples from cancer patients and control individuals. Receiver-operating characteristic analyses determined the cutoff value of CEA for NSCLC detection to be 2.5 ng/mL (with a sensitivity of 39.3% and a specificity of 94.6%) and also determined the cutoff value of CYFRA 21-1 to be 2.0 pg/mL (with a sensitivity of 33.9% and a specificity of 97.2%; data not shown). As shown in Supplementary Fig. S1A (left and middle), the correlation coefficient between serum CEA and CYFRA 21-1 values was significant (Spearman rank correlation {rho} = 0.355, P = 0.0002), whereas the correlation between serum LY6K and CEA values was not significant (Spearman rank correlation {rho} = 0.021, P = 0.8275), indicating that measuring both markers in serum can improve overall sensitivity for detection of NSCLC to 61.6%. False-positive results for either of the two tumor markers among normal volunteers (control group) accounted for 9.5%, whereas the false-positive rates for CEA and LY6K in the same control group were 4.1% and 5.4%, respectively. According to tumor histology, the sensitivity of the combination of serum LY6K and CEA as a tumor detection marker was 64.7% for adenocarcinoma and 51.6% for SCC, suggesting the usefulness of this combination for adenocarcinoma detection.

The correlation coefficient between serum LY6K and CYFRA 21-1 values for NSCLC patients was not significant (Spearman rank correlation {rho} = 0.119, P = 0.2114; Supplementary Fig. S1A, right panel), also indicating that measurement of serum levels of both markers can improve overall sensitivity for detection of NSCLC to 59.8%; for diagnosing NSCLC, the sensitivity of CYFRA 21-1 alone was 33.9%. False-positive cases for either of the two tumor markers among normal volunteers (control group) were 6.8%, although the false-positive rates for CYFRA 21-1 in the same control group were 2.7%. According to tumor histology, the sensitivity of the combination of serum LY6K and CYFRA 21-1 for the detection of tumors was 56.5% for adenocarcinoma and 70.4% for SCC, indicating the usefulness of this combination for SCC detection. Combination of LY6K with both CEA and CYFRA 21-1 indicated that 21 of 54 (38.9%) NSCLC patients who were negative for both CEA and CYFRA 21-1 were diagnosed as LY6K-positive (Supplementary Fig. S1B).

We further measured by ELISA serum levels of CEA and CYFRA 21-1 in the same set of serum samples from ESCC patients (Supplementary Fig. S1C). The correlation coefficient between serum LY6K and CEA values for ESCC patients was not significant (Spearman rank correlation {rho} = 0.153, P = 0.0781; Supplementary Fig. S1C, middle), indicating that measuring both markers in serum can improve overall sensitivity for detection of ESCC to 44.3% whereas the sensitivity of CEA alone was 18.0%. The correlation between serum LY6K and CYFRA 21-1 values for ESCC patients was also not significant (Spearman rank correlation {rho} = 0.034, P = 0.6989; Supplementary Fig. S1C, right). A combined assay for both LY6K and CYFRA 21-1 classified 52.5% of ESCC patients as positive, whereas the sensitivity of CYFRA 21-1 alone was 23.0%. Combination of LY6K with both CEA and CYFRA 21-1 indicated that 16 of 40 (40.0%) ESCC patients who were negative for both CEA and CYFRA 21-1 were diagnosed as LY6K-positive (Supplementary Fig. S1D). The data clearly suggest that serum LY6K levels were also high in certain proportion of cancer patients that could not be diagnosed by the combination of CEA and CYFRA 21-1.

Effect of LY6K-siRNAs on growth of NSCLC cells. To assess whether LY6K plays a role in growth or survival of lung cancer cells, we designed and constructed plasmids to express siRNA against LY6K (si-LY6K-1 and si-LY6K-2), along with two different control plasmids (siRNAs for EGFP and SCR), and transfected them into lung cancer (RERF-LC-AI and LC319) and esophageal cancer (TE8) cells to suppress expression of endogenous LY6K (representative data of RERF-LC-AI and TE8 was shown in Fig. 4 and Supplementary Fig. S2). The amount of LY6K protein in the cells transfected with si-LY6K-2 was significantly decreased in comparison with cells transfected with any of the two control siRNAs or si-LY6K-1 (Fig. 4 and Supplementary Fig. S2). In accordance with its suppressive effect on protein levels of LY6K, transfected si-LY6K-2 caused significant decreases in the numbers of viable cells (Fig. 4 and Supplementary Fig. S2).


Figure 4
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  		Figure 4. Growth inhibition of NSCLC cells by siRNA against LY6K. Response of RERF-LC-AI cells to si-LY6K-1 and si-LY6K-2 or control siRNAs (EGFP or SCR). Left top, the level of LY6K protein expression detected by Western blot analysis in cells treated with either control or si-LY6Ks. Left bottom, colony formation assays using RERF-LC-AI cells transfected with si-LY6K-1 and si-LY6K-2, si-EGFP, or si-SCR. Right, the effect of siRNA against LY6K on cell viability, detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. All assays were performed thrice and in triplicate wells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent acceleration in identification and characterization of novel molecular targets for cancer therapy has enhanced development of new types of anticancer agents or antibodies and cancer vaccines (8). Molecular targeted drugs are expected to be highly specific to malignant cells, with minimal adverse effects due to their well-defined mechanisms of action. As an approach to such goal, one promising strategy is to combine the power of genome-wide expression analysis to effectively screen genes that are overexpressed in cancer cells but scarcely expressed in normal organs, with high throughput screening of their protein expression related to clinical outcome by means of tissue microarray, as well as with examining loss of function phenotypes by RNA interference systems (19–33). Using this combination of approaches for the study, we have shown that LY6K is a novel cancer-testis antigen, whose overexpression shows unfavorable prognostic significance in NSCLC patients and affects on growth of the cancer cells.

Like other LY6 antigens, LY6K has 10 cysteine residues in a conserved position and harbors the sequence structure that theoretically determines GPI anchoring. Members of the LY6 family are suspected to have functions related to cell signaling and/or cell adhesion (41), although the precise role of LY6K in lung carcinogenesis or the physiologic function in the normal cells is unknown. Because LY6K gene is located at chromosome 8q24, a region of allelic gain in more than half of lung cancers (42), its overexpression might be partly explained by amplification or chromosomal aberration at this locus.

There are several known GPI-anchored proteins that are applicable to diagnosis of human cancer in certain clinical or preclinical settings (43). Human CEA is supposed to be a GPI-anchored protein (43) and expressed highly in a significant proportion of relatively advanced adenocarcinomas, particularly those from the colon, pancreas, breast, and lung (44). Its presence in serum in cancer patients has been used for disease staging and as an indicator of residual disease and/or tumor recurrences (44). In addition, some tumor-specific markers and prognostic markers, such as CD109, glypican-3, CEA-related cell adhesion molecule 6, and prostate stem cell antigen, are categorized as the GPI-anchored protein (40, 45–47). Among them, CD109 and glypican-3 are also known to be the cancer-testis antigens. On the other hand, there are several reports on GPI-anchored proteins as immunotherapeutic targets for human cancer. The CEA-TRICOM (three T-cell costimulatory molecules: B7-1, intercellular adhesion molecule (ICAM)-1, and LFA-3, which were designated TRICOM) vaccines has been shown to safely generate significant CEA-specific immune responses against advanced cancer in the phase I clinical trial (48). Recently, two independent studies showed that a passive immunotherapy approach using anti–prostate stem cell antigen monoclonal antibody inhibited prostate tumor growth and metastasis formation and further prolonged survival times of mice bearing human prostate cancer xenografts (49, 50).

In this study, we showed that LY6K was expressed only in testis among the normal tissues examined and was highly expressed in 88.2% of surgically resected samples from NSCLC patients and in 95.1% of those from ESCC patients. The LY6K overexpression was associated with the shorter cancer-specific survival period. Suppression of LY6K expression with siRNA effectively suppressed growth of lung and esophageal cancer cells that expressed LY6K. The combined results strongly suggest that LY6K is likely to be associated with highly malignant phenotype of those tumors. Because LY6K is considered to be the cancer-testis antigens, LY6K might be a good target for cancer immunotherapy.

We also found that LY6K protein was secreted into serum from patients with lung cancer or esophageal cancer that strongly expressed LY6K. Positivity of serum LY6K seems to be considerably correlated with the presence of primary tumors, because the concentration of serum LY6K was dramatically reduced after surgical resection of primary tumors and the levels of serum LY6K showed good correlation with the expression levels of LY6K in primary tumor tissue in the same patients. Interestingly, the correlation coefficient between serum LY6K and CEA or CYFRA 21-1 values was not significant, whereas the correlation coefficient between serum CEA and CYFRA 21-1 values was significant. In fact, 38.9% to 40.0% of NSCLC and ESCC patients who were negative for both CEA and CYFRA 21-1 were diagnosed to be positive for LY6K (Supplementary Fig. S1B and D). An assay combining both LY6K and CEA/CYFRA 21-1 increased the sensitivity, such that 64.7% to 70.4% of the patients with NSCLC and 52.5% of ESCC were diagnosed as positive whereas 6.8% to 9.5% of healthy volunteers were falsely diagnosed as positive. On the other hand, the sensitivity of the combination of conventional serum tumor marker, CEA and CYFRA 21-1 in the same set of serum samples, was 51.8% for NSCLC (53.0% for adenocarcinoma and 48.1% for SCC) and 34.4% for ESCC, whereas false-positive cases for either of the two tumor markers among normal volunteers (control group) were 6.8% (Supplementary Fig. S1B and D). Although additional validation with a larger set of serum samples covering various clinical stages will be necessary, the data presented here sufficiently show a potential clinical application of LY6K itself as a serologic/histochemical biomarker for lung and esophageal cancers. It should also be noted that we observed activation of LY6K in more than half of a series of other types of cancers, such as cervical carcinomas (data not shown), suggesting its diagnostic and therapeutic application to a wide range of tumors.

In summary, we have shown LY6K cancer-testis antigen as a potential biomarker for diagnosis of lung and esophageal cancers, as well as for monitoring patients with these diseases. LY6K, combined with other tumor markers, could significantly improve the sensitivity of cancer diagnosis, whereas it could be used at initial diagnosis to identify patients who might benefit from early systemic treatment. Moreover, LY6K is likely to be an essential contributor to aggressive features of NSCLC and ESCC and a likely target for development of therapeutic approaches, such as molecular targeted drugs and immunotherapy to any types of cancers overexpressing this molecule.


   Acknowledgments
 
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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 8/22/07. Revised 10/17/07. Accepted 10/22/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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