Cancer-Testis Genes Are Coordinately Expressed and Are Markers of ...

Imaging, Diagnosis, Prognosis

Cancer-Testis Genes Are Coordinately Expressed and Are Markers of Poor Outcome in Non ^ Small Cell Lung Cancer Ali O. Gure,1 Ramon Chua,1 Barbara Williamson,1 Mithat Gonen,2 Cathy A. Ferrera,3 Sacha Gnjatic,1 Gerd Ritter,1 Andrew J.G. Simpson,1 Yao-T. Chen,4 Lloyd J. Old,1 and Nasser K. Altorki3

Abstract

Purpose: Cancer-testis genes mapping to the X chromosome have common expression patterns and show similar responses to modulators of epigenetic mechanisms.We asked whether cancertestis gene expression occurred coordinately, and whether it correlated with variables of disease and clinical outcome of non ^ small cell lung cancer (NSCLC). Experimental Design:Tumors from 523 NSCLC patients undergoing surgery were evaluated for the expression of nine cancer-testis genes (NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7/MAGE-C1, SSX2, and SSX4) by semiquantitative PCR. Clinical data available for 447 patients were used to correlate cancer-testis expression to variables of disease and clinical outcome. Results: At least one cancer-testis gene was expressed by 90% of squamous carcinoma, 62% of bronchioloalveolar cancer, and 67% of adenocarcinoma samples. Statistically significant coexpression was observed for 34 of the 36 possible cancer-testis combinations. Cancer-testis gene expression, either cumulatively or individually, showed significant associations with male sex, smoking history, advanced tumor, nodal and pathologic stages, pleural invasion, and the absence of ground glass opacity. Cox regression analysis revealed the expression of NY-ESO-1and MAGEA3 as markers of poor prognosis, independent of confounding variables for adenocarcinoma of the lung. Conclusions: Cancer-testis genes are coordinately expressed in NSCLC, and their expression is associated with advanced disease and poor outcome.

Cancer-testis gene expression is observed at different frequencies in all tumors regardless of tissue of origin. Of the 40 or so cancer-testis genes/gene families, more than half map to chromosome X (http://www.cancerimmunity.org/CTdatabase/). These X chromosome cancer-testis genes (CT-X) and BAGE genes that map to juxtacentromeric regions are typically multigene families that have arisen through chromosomal duplications. Most cancer-testis genes have been initially identified through immunologic assays (1). In normal tissues, CT-X genes are consistently expressed in spermatogonia, oogonia, and the trophoblast cells (1). In testicular germ cells, the expression of most CT-X genes decrease as cells enter Authors’ Affiliations: 1Ludwig Institute for Cancer Research, 2Department of Statistics and Epidemiology, Memorial Sloan Kettering Cancer Center ; Departments of 3Cardiothoracic Surgery and 4Pathology, Weill Medical College of Cornell University, New York, New York Received 6/2/05; revised 8/8/05; accepted 8/23/05. Grant support: Ludwig Institute for Cancer Research. 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. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Ali O. Gure, Ludwig Institute for Cancer Research, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10021. Phone: 212-746-6450; Fax: 212-746-4483; E-mail: agure@ med.cornell.edu. F 2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1203

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meiosis (2), and regain genomic methylation (3, 4), coinciding with the loss of both Suv39h2 expression and H4 hyperacetylation (5, 6). Cancer-testis genes mapping to somatic chromosomes, on the other hand, tend to have fewer homologues and can be expressed in meiotic gametes (7 – 9). Based on these differences and the common epigenetic mechanisms associated with the regulation of their expression, it has been proposed that CT-X genes might constitute a distinct group (10). Ectopic hypomethylation of genomic DNA has been associated with CT-X gene expression and the demethylation of critical CpG residues within their promoter regions (11). All CT-X genes that are expressed in tumors or testis can be induced in vitro by DNA demethylation or by inhibitors of histone deacetylation (1). Despite the evidence suggesting that CT-X genes share common epigenetic regulatory mechanisms, the evidence regarding CT-X coexpression and whether CT-X expression associates with clinical variables of disease and outcome is inconclusive. In this study, we analyzed tumors from 523 non – small cell lung cancer (NSCLC) patients for the expression of nine CT-X genes. We show coordinate expression among all CT-X genes tested. In 447 patients for whom clinical data were available, we evaluated the association of CT-X expression with variables of disease and outcome. CT-X expression was found to be sporadically associated with advanced disease and other variables that indicate worse prognosis in all major histologic types of NSCLC. In addition, Cox regression analysis revealed

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Clin Cancer Res 2005;11(22) November 15, 2005

Imaging, Diagnosis, Prognosis

Total RNA was prepared following homogenization by the guanidium isothiocyanade method followed by CsCl gradient centrifugation. Alternatively, the Ribopure kit (Ambion, Austin, TX) was used according to manufacturer’s instructions. Remaining tumor tissue was maintained either frozen or in RNAlater (Ambion). Total RNA (2 Ag) was reverse-transcribed with 200 units Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions, in the presence of 2 Ag random hexamers (Applied Biosystems, Foster City, CA), 20 units RNaseOUT (Invitrogen), and 5 mmol/L DTT in a total volume of 20 AL. Each reverse transcription reaction was done in the presence or absence of Moloney murine leukemia virus-RTase to test for contamination. For individual PCR reactions, 250 ng of cDNA were amplified with gene-specific oligonucleotides (2 ng per 25 AL reaction) in the presence of 1 unit AmpliTaq Gold (Applied Biosystems) and 5 Amol/L of each deoxynucleotidetriphosphates (Applied Biosystems). Gene-specific primers used to amplify individual CT-X transcripts are shown in Supplementary Table S1. The integrity of cDNA obtained was tested by amplification of p53 transcripts (p53-F, 5V-TACTCCCCTGCCCTCAACAAG; p53-R, 5V-CTCAGGCGGCTCATAGGG). For semiquantitative PCR analysis, reverse transcription-PCR products were categorized after separation on ethidium bromide – stained agarose gels as either , +/ , +, ++, or +++, reflecting the intensity of the product when compared with a standardized testis sample (Fig. 1). Real-time reverse transcription-PCR analysis (ABI Prism, Applied Biosciences) of a representative number of tumor RNA suggested the different intensities corresponded to f0.1-1 fg (+/ ), 1-5 fg (+), 5-100 fg (++), and 0 indicate agreement better than chance. For example, j = 0.5 means that the expression of two given CT-X genes occurs simultaneously 50% of the time over and above that expected by chance alone (14). To evaluate whether cancer-testis gene expression was related to sex, smoking status, tumor size, pleural invasion, disease stage, and other clinical variables, univariate analysis using the Wilcoxon rank sum test

Fig. 1. Semiquantitative RT-PCR. Examples of reverse transcription-PCR (RT-PCR) reactions resulting in different quantities of products ( to +++). Tumor samples are indicated by numbers. T, testis. Control amplifications without reverse transcriptase (RT ). p53 amplification was done to ensure RNA integrity. The larger p53 band in lanes without RT is due to amplification of contaminating genomic DNA.

that expression of NY-ESO-1 and MAGE-A3 were independent markers of worse outcome in adenocarcinomas of the lung. CT-X expression was not found to affect outcome in squamous carcinomas, bronchioloalveolar carcinoma, or adenocarcinoma with bronchioloalveolar features.

Patients and Methods Patients. A total of 523 patients undergoing curative surgical resection for primary NSCLC at the Department of Cardio-Thoracic Surgery, Weill Medical College of Cornell University, from 1991 to July 2004, were included in this study. Informed consent was obtained from all patients. The study was approved by the Institutional Review Board of Weill Medical College of Cornell University. Expression analysis. Tumor tissues were obtained during surgery. Following gross dissection, tissues were immediately frozen on dry ice.

Table 1. Frequency of CT-X gene expression in NSCLC All histologies

Any CT-X NY-ESO-1 LAGE-1 MAGE-A1 MAGE-A3 MAGE-A4 MAGE-A10 CT7 SSX2 SSX4

+

Adenocarcinoma +

BAC + AdenoBAC

n

CT (%)

CT[high] (%)

n

CT (%)

CT[high] (%)

n

523 518 443 475 520 314 250 323 231 215

72 27 32 46 45 35 27 18 10 14

49 15 20 30 40 22 19 10 5 7

221 220 183 200 219 118 84 147 102 89

67 22 27 41 46 29 25 16 9 9

42 14 19 26 27 15 19 8 4 5

95 95 83 90 95 75 73 57 52 47

+

CT (%)

62 18 31 34 43 16 15 12 2 6

CT[high] (%) 34 5 11 19 27 5 10 5 0 2

Squamous cell carcinoma +

Other

n

CT (%)

CT[high] (%)

n

97 95 85 87 96 57 42 58 48 42

90 43 45 71 80 61 50 28 10 19

70 19 33 52 65 49 41 17 8 10

110 108 92 98 110 64 51 61 29 37

CT+ CT[high] (%) (%) 76 30 32 46 64 44 29 20 24 27

56 19 19 30 48 31 16 12 14 14

Abbreviations: BAC, bronchioloalveolar cancer; AdenoBAC, adenobronchioloalveolar cancer; CT, cancer-testis.

Clin Cancer Res 2005;11(22) November 15, 2005

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Cancer-Testis Genes: Markers of Poor Outcome in NSCLC (or t test) and Fisher’s exact test (or m2 test) were used for continuous and categorical variables, respectively. The effect of cancer-testis gene expression on survival was evaluated using the Kaplan-Meier method, and differences between two groups were compared using the log-rank test. All survival curves were calculated from the date of surgery. All statistical analyses were two sided with a 5% type I error rate and were computed using SAS (version 9.0) software (SAS Institute, Cary, NC). P < 0.05 was considered statistically significant. Covariates with P < 0.05 by univariate analysis were subjected to multivariate analysis. Cox regression analysis was done to assess the effects of CT-X expression on survival while controlling for confounding clinical covariates.

Results Frequency of X chromosome cancer-testis genes in non – small cell lung cancer. A total of 523 cases of NSCLC were typed for

CT-X expression. The comparative level of CT-X expression was estimated by semiquantitative PCR and recorded as +/ , +, ++, and +++ based on the intensity of the amplification product (Fig. 1). Among tumors tested, 377 (72.1%) expressed at least one of the nine CT-X genes tested. The most frequently observed CT-X was MAGE-A3, present in 55.2% of samples followed by MAGE-A1 (46.3%), MAGE-A4 (34.7%), LAGE-1 (32.1%), MAGE-A10 (27.2%), NY-ESO-1 (26.6%), MAGE-C1/ CT7 (18.8%), SSX4 (13.5%), and SSX2 (9.6%; Table 1). The expression frequency of CT-X genes showed striking differences between histologic subtypes. Sixty-two percent (59 of 95) of the bronchioloalveolar carcinoma cases (including adenocarcinomas with bronchioloalveolar features) expressed at least one CT-X gene. This frequency was 67% (148 of 221) for adenocarcinomas and 90% (87 of 97) for squamous cell

Table 2. Coexpression of CT-X genes in NSCLC CT-X1

CT-X2

NY-ESO-1 NY-ESO-1 NY-ESO-1 NY-ESO-1 NY-ESO-1 NY-ESO-1 NY-ESO-1 NY-ESO-1 LAGE-1 LAGE-1 LAGE-1 LAGE-1 LAGE-1 LAGE-1 LAGE-1 MAGE-A1 MAGE-A1 MAGE-A1 MAGE-A1 MAGE-A1 MAGE-A1 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A4 MAGE-A4 MAGE-A4 MAGE-A4 MAGE-A10 MAGE-A10 MAGE-A10 CT7 CT7 SSX2

LAGE-1 MAGE-A1 MAGE-A3 MAGE-A4 MAGE-A10 CT7 SSX2 SSX4 MAGE-A1 MAGE-A3 MAGE-A4 MAGE-A10 CT7 SSX2 SSX4 MAGE-A3 MAGE-A4 MAGE-A10 CT7 SSX2 SSX4 MAGE-A4 MAGE-A10 CT7 SSX2 SSX4 MAGE-A10 CT7 SSX2 SSX4 CT7 SSX2 SSX4 SSX2 SSX4 SSX4


Samples tested for both CT-X1 and CT-X2

% Samples expressing CT-X1 among CT-X2(+) samples

% Observed/ % expected ratio for CT-X1 among CT-X2 (+) samples

% Samples expressing CT-X2 among CT-X1(+) samples

% Observed/ % expected ratio for CT-X2 among CT-X1 (+) samples

k

P

442 474 516 313 249 323 229 214 409 442 302 250 269 189 174 473 309 250 322 209 194 313 250 321 228 213 247 218 181 162 166 142 128 136 108 194

57 44 40 49 53 51 68 72 51 45 49 54 56 75 56 70 72 93 71 90 88 76 91 90 91 97 76 55 72 75 54 72 68 56 36 35

2 1.7 1.5 1.8 1.8 2.1 2.3 2.1 1.7 1.5 1.4 1.6 1.9 2.1 1.4 1.5 1.4 1.8 1.6 1.9 1.6 1.3 1.7 1.7 1.7 1.7 2.3 1.6 1.8 1.9 2 2.6 2.4 2.8 1.5 3.5

64 78 83 63 51 40 22 29 80 81 51 44 37 22 21 84 49 48 30 18 22 47 43 31 16 23 62 28 18 28 34 25 36 19 19 50

2 1.7 1.5 1.8 1.9 2.2 2.2 2.1 1.6 1.4 1.8 1.6 2 2.2 1.3 1.5 1.4 1.8 1.7 1.8 1.7 1.3 1.6 1.7 1.6 1.6 2.3 1.7 1.8 1.9 2 2.5 2.4 2.1 1.5 3.3

0.43 0.34 0.28 0.36 0.33 0.30 0.23 0.27 0.39 0.27 0.24 0.26 0.28 0.22 0.11 0.52 0.30 0.43 0.22 0.16 0.18 0.26 0.33 0.26 0.13 0.19 0.55 0.18 0.15 0.25 0.27 0.27 0.35 0.10 0.10 0.33