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Cyclin D1 Guanine/Adenine 870 Polymorphism With Altered Protein Expression Is Associated With Genomic Instability and Aggressive Clinical Biology of Esophageal Adenocarcinoma

Julie G. Izzo, Tsung-Teh Wu, Xifeng Wu, Joe Ensor, Rajyalakshmi Luthra, Jennifer Pan, Arlene Correa, Stephen G. Swisher, Clifford K.S. Chao, Walter N. Hittelman, Jaffer A. Ajani

From the Departments of Experimental Therapeutics, Pathology, Biostatistics and Applied Mathematics, Thoracic and Cardio-Vascular Surgery, Radiation Oncology, Epidemiology, and Gastrointestinal Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX

Address reprint requests to Julie G. Izzo, MD, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Unit 19, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: jizzo{at}mdanderson.org

	ABSTRACT

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PURPOSE: Altered cyclin D1 (CD1), a cell cycle regulator, may play an important role in imparting aggressive nature to esophageal adenocarcinoma (EAC). CD1 gene single nucleotide polymorphism G/A870 results in two alternatively spliced transcripts, CD1a and CD1b. CD1b, preferentially encoded by the A870 allele, is putatively oncogenic. We hypothesized that CD1 A870 allele would be associated with higher CD1 protein expression, and increased genomic instability during EAC evolution, leading to more aggressive phenotype.

PATIENTS AND METHODS: One hundred twenty-four archival specimens of EAC, and 39 associated Barrett's esophagus (BE) specimens were examined for CD1 genotype, CD1 protein expression, and chromosome 9 polysomy (representing genomic instability). We correlated CD1 genotypes with CD1 protein expression, genomic instability, age at diagnosis of EAC, and overall survival (OS).

RESULTS: The A870 allele was associated with higher levels of CD1 protein expression in EAC (P = .032); in BE (P = .01) where it was associated with concomitant increased chromosome 9 polysomy (P = .002); and with a younger age at diagnosis (P < .001) and poor OS (P = .0003) of EAC patients.

CONCLUSION: Our data suggest that CD1 A870 background may be imparting aggressive phenotype to EAC. It provides a molecular basis to explain the clinical biology associated with CD1 polymorphism whereas aberrant nuclear accumulation of CD1 protein enhances the acquisition of genomic instability (ie, clonal diversity), thus leading to early age of EAC diagnosis and poor OS. CD1 genotyping with other biomarkers may help create a biomarker-based prognostic model for EAC and CD1 may also serve as a therapeutic target.

	INTRODUCTION

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Marked increase in the incidence and poor overall survival rates of esophageal adenocarcinoma (EAC)¹ underscore the need for molecular markers that might become useful for risk assessment, early diagnosis, prognosis, and targeted therapeutic intervention.

Carcinogenesis is a multistep process driven by genetic instability with the emergence of genetically altered clones that are fit to survive, expand, and metastasize.^2-4 Cytogenetic and molecular analyses of EAC and of associated premalignant lesions, such as Barrett's esophagus (BE), have identified a variety of common epigenetic and genetic changes.^5-8 However, predictive and prognostic biomarkers for EAC are mostly lacking.

Alterations of cyclin D1 (CD1) are considered potential biomarkers for EAC and its progression.^9-12 CD1 is an important regulatory protein for the G1-S cell cycle phase transition, and for transcriptional control of mammalian cells, in which CD1 participates to the regulation of proliferation and differentiation.¹³ In healthy cells, the expression of CD1 is strictly regulated by a cascade of intracellular events.^13,14 In organized epithelia, downregulation of CD1 expression is necessary for ordered differentiation.¹⁵ The proper balance of proliferation and differentiation is crucial for tissue homeostasis, but is altered in premalignant and malignant conditions. Deregulated CD1 promotes genetic instability in vitro and tumorigenesis in vivo.^16-18 Both, CD1 gene alterations and/or protein deregulation have been widely observed in human malignant and premalignant tissues, including EAC and BE. ^9-11,19-21

Both in EAC and BE, CD1 overexpression has been frequently found (up to 46% and 64%, respectively).^9-11,20,21 In BE, CD1 overexpression has been proposed as an end point biomarker for cancer development⁹; however, its impact on the clinical biology of EAC has not been established. In addition, the low frequency of CD1 gene amplification in EAC²¹ suggests that other mechanisms may be implicated in CD1 protein deregulation.

Several groups, including us, have investigated the functional significance of a guanine (G)/adenine (A) single nucleotide polymorphism at position 870 of the CD1 gene, which is associated with two different splice-variant transcripts.^22-27 The normally spliced variant includes exon 5, which carries a destruction box sequence, responsible for nuclear to cytoplasmic export and consequent ubiquitin-mediated proteolysis.^13,22 The alternatively spliced transcript encodes for a protein lacking the ubiquitin destruction box, leading to increased nuclear half-life of the protein.^28-30 Both the A and G alleles can encode for the two transcripts;³¹ however, the A allele preferentially encodes the alternate transcript, CD1b, leading to increased levels of nuclear CD1 even in the heterozygote state. While its biologic functions have not been completely elucidated, the CD1 alternate form has been shown in vitro to be a nuclear oncogene.³² A few studies have analyzed the relationship of CD1 genotype and genetic susceptibility for EAC.^33-36 However, none of these studies investigated the relationship of CD1 G/A870 polymorphism and its associated protein expression with genomic instability, age at diagnosis of EAC, and prognosis of patients with EAC.

To better understand the functional impact of CD1 G/A870 polymorphism in defining the clinical biology of EAC, we conducted a biomarker study in archival tissues containing EAC and BE lesions adjacent to EAC.

	PATIENTS AND METHODS

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Patients
One hundred twenty-four patients who underwent esophagectomy as primary treatment of EAC between January 1986 and December 1997 at our institution were included. All patients were clinically staged using barium-swallow radiography and computed tomography of chest, abdomen, and esophagoscopy. Transthoracic or transhiatal approaches were used. None of the patients received neoadjuvant chemoradiation treatments; four patients received fluorouracil-based adjuvant chemotherapy. Pathologic restaging was performed according to International Union Against Cancer.³⁷ Postsurgical surveillance was performed every 3 months during the first year, every 6 months for 2 additional years, then yearly. This study was approved by the institutional review board.

Tissue Specimens
All specimens were obtained retrospectively and underwent pathologic diagnosis at the time of surgery. Hematoxylin-eosin stained slides were re-reviewed by one gastrointestinal experienced pathologist (T.T.W.). Subsequently, unstained slides made from formalin-fixed paraffin-embedded (FFPE) blocks were cut into 4 µm thick sections and adjacent sections were used for biomarker assessment by immunohistochemistry and fluorescent in situ hybridization (FISH). EAC were available for 124 patients. BE, either adjacent to EAC or distant in the esophagectomy specimen, was available in 39 of 124 patients. Twenty three of the 39 patients had one histologic lesion type (metaplasia or low-grade or high-grade dysplasia), 17 patients had two histologic lesions, and four patients had all three histologic lesions.³⁸ BE histologic lesions included: 30 metaplasia, 12 low-grade dysplasia, and 16 high-grade dysplasia.

CD1 Genotype Assessment
DNA single-strand conformation polymorphism and DNA sequencing analyses were performed to determine CD1 genotype, as previously reported.²⁶ All single-strand conformation polymorphism assays were repeated twice, and all were sequenced. FFPE sections containing healthy mucosa were used to obtain DNA after microdisssection, as previously described.³⁹ Heterozygote (AG) human placental DNA was included as positive control, while DNA was omitted in negative control samples. CD1 polymorphism was evaluated independently, in blinded fashion by three investigators (J.G.I., J.P., I.M.B.) without knowledge of CD1 protein expression, chromosome polysomy, and clinical data.

CD1 Immunohistochemistry
CD1 analysis was performed on FFPE sections with a mouse monoclonal antibody (clone P2D11F11; Novocastra, Newcastle, United Kingdom), as previously reported.^26,40 This antibody recognizes both CD1 protein variants.^22,40 Only nuclear immunoreactivity was considered positive. The intensity of nuclear staining was evaluated as: 0 = no staining, 1 = weak, 2 = moderate, or 3 = strong. FFPE cell pellets of MDA-1986 cancer cell line, strongly expressing CD1,⁴¹ were placed on each section as positive control. Negative controls were included in each immunohistochemistry batch and consisted in a slide of FFPE MDA-1986 immunolabeled without primary antibody. Lymphocytes residing in each tissue specimen served as internal negative controls. For comparison with patient characteristics and clinical outcome, the results were expressed as the labeling index (LI), that is, the fraction of CD1 positive cells expressing intensity 2 or 3. Deregulated CD1 was defined as LI 10%, based on the distribution of expression observed in the lesions examined and similar cutoffs previously chosen.^20,26 CD1 protein levels were described using weighted mean index (WMI), which is the sum of the intensity scale values (0 to 3) of each cell divided by the total number of evaluated cells.²⁶ All assessable cells in EAC and BE were included in the analysis. Three investigators (J.G.I., J.P., T.T.W.) determined CD1 immunolabeling in blinded fashion, without knowledge of genotype, chromosome polysomy, and clinical data. In discrepant cases, a final opinion was made based on consensus by all three investigators.

Chromosome Instability Analysis
The degree of chromosome instability was assessed by measuring chromosome 9 copy number using FISH on FFPE sections, as previously described.²⁶ Chromosome 9 was chosen because polysomy for chromosome 9 correlates with other chromosome polysomies.⁴² Directly labeled DNA chromosomes 9 probes (Vysis, Downers Grove, IL) were used. FISH signals were visualized with a Zeiss Axioplan 2 epifluorescent microscope (Zeiss, Thornwood, NY), equipped with a high-resolution image analysis system (MetaMorph, Universal Imaging, Downington, PA). Two investigators (J.G.I., J.P.) were blinded to evaluation of the FISH signals and used criteria previously described.⁴³ All assessable nuclei were analyzed per section and histologic area. The chromosome polysomy index (CPI) was defined as the percentage of scored nuclei exhibiting three or more chromosome copies.⁴⁴ A 5% cutoff point was used to stratify tissues in low and high CPI groups. This cutoff was based on the distribution of chromosomes indices observed in this population, the median being 4.3% (range, 0% to 20%) and the mean ± standard deviation being 0.061 ± 0.038.

Statistical Analysis
The analysis of association between clinicopathologic variables and CD1 polymorphism was conducted separately for each CD1 genotype (AA, AG, GG) using Fisher's exact, Wilcoxon rank sum, and Cochran-Armitage trend tests. The ² goodness of fit test was used to test for the Hardy-Weinberg equilibrium analysis. The age at cancer diagnosis and overall survival for each CD1 genotype was estimated by the Kaplan-Meier method; the log-rank test was used for statistical significance. Overall survival was defined as the time from diagnosis to death. Perioperative mortality, defined as death within 30 days postsurgery, was excluded from the survival analyses because the end point of the study was long-term survival and not short-term morbidity due to surgery. After stepwise selection to determine which covariate (CD1 genotypes, age at diagnosis, clinical stage, tumor location, histology parameters, surgical technique) was a significant predictor of overall survival, multivariate Cox proportional hazards models were adjusted for potential demographic confounding factors and fit to yield ratio estimates. Because of the biologic functional dominant effect of the A allele, the analysis to determine associations between CD1 genotype, and biologic variables was conducted for two CD1 genotypes groups (ie, GG and combined AA/AG group). The Spearman rank r test was performed to test for bivariate relationships between CD1 genotype, protein expression, and chromosome polysomy. All statistical analyses were two sided and considered significant if P < .05. The computations were carried out using the SAS software, version 6.12 (SAS Institute Inc, Cary, NC).

	RESULTS

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Patients Characteristics
Of 124 patients' specimens examined, twenty three carried the AA (18.5%), 54 carried the AG (43.5%), and 47 carried the GG (38%) genotypes, as previously reported.⁴⁵ The calculated allele frequency distributions were 40.3% for the A allele and 59.7% for the G allele. The observed allele and genotype frequencies did not show significant deviation from the Hardy-Weinberg equilibrium (², 2.8, two degrees of freedom; P > .05) and were similar to those found in healthy controls previously studied at our institution, and in patients with esophageal cancers.^23-25,33-36 The patient characteristics at diagnosis are summarized in Table 1. The CD1 genotype was not statistically associated with sex, race, cancer stage, histologic characteristics, surgical technique, or postsurgical residual tumor. The age of EAC diagnosis for the different CD1 genotypes using Kaplan-Meier survival showed that the AA and AG genotype patients developed cancer at a significantly younger age compared with the GG group (log-rank test, P < .0001; Fig 1). There was no difference between the average age at diagnosis between the AA (median, 59; range, 42 to 77 years) and AG groups (median, 59; range, 28 to 79 years). However, the GG group had significantly older age at diagnosis (median, 69; range, 44 to 85 years) with a 10.5-year difference compared with the A allele bearing genotypes (Wilcoxon test P < .001). Based on the similar clinical phenotype observed for AA and AG genotypes and because the A allele can have a functional dominant effect on the half-life of the CD1 protein, the AA and AG genotypes were combined for biologic-related analyses.

View larger version (19K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Kaplan-Meier analysis of age of cancer diagnosis for subjects with the AA, AG, and GG genotypes. N and S indicate, respectively, the number of patients free of cancer and the Kaplan-Meier estimate of cancer diagnosis at age 40, 50, 60, 70, and 80. O, event indicator; A, adenosine; G, guanine.

View larger version (55K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. CD1 protein expression detected by immunohistochemistry. (A) AA cancer at 20x magnification. Top inset of (A): invasive tumor front bottom inset: (40x magnification); bottom inset of (A): tumor core (40x magnification). (B) GG cancer at 20x magnification; inset for (B): weak nuclear staining (40x magnification). A, adenosine; G, guanine.

View larger version (18K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Overall survival for AA, AG, and GG genotype patients with esophageal adenocarcinoma. (A) Kaplan-Meier estimates and plot; (B) multivariate analysis. (*) Adjusted for sex and race. () 2 analysis comparing the variables in the multiple regression setting. O, event indicator; +, censoring indicator; HR, hazard ratio; A, adenosine; G, guanine.

CD1 Genotype and Protein Expression in BE
Of 39 patients with available BE specimens associated with EAC, 10 had AA genotype (26%), 19 had AG genotype (49%), and 10 had GG genotype (26%). Deregulated CD1 was found in all BE specimens, however, both labeling and WMIs were higher in BE with AA/AG genotype compared with GG genotype (LI median, AA/AG = 0.6; range, 0.46 to 0.82 v GG = 0.3; range, 0.20 to 0.54; Wilcoxon test P = .09; WMI median, AA/AG = 1.03; range, 0.40 to 1.54 v GG = 0.16; range, 0.09 to 0.51; Wilcoxon test P = .01).

Examination of CD1 labeling indices in each BE histologic subtype (ie, metaplasia, low-grade and high-grade dysplasia) revealed that the AA/AG group had increased number of CD1 positive cells in all three lesions (Table 1). Moreover, the AA/AG genotype had also significantly higher nuclear CD1 protein levels by WMI, compared with GG genotype in all three categories (Table 2).

As illustrated in Figures 4, 5, and 6, BE with AA/AG genotype had increased CD1 labeling throughout the whole BE crypts, suggesting a defect in CD1 downregulation and turnover away from the proliferating compartment.

View larger version (103K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 4. AA/AG Barrett's esophagus metaplasia. (A) CD1 protein expression by immunohistochemistry; (B) hematoxylin-eosin stain.

View larger version (105K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 5. AA/AG Barrett's esophagus low-grade dysplasia. (A) CD1 protein expression by immunohistochemistry; (B) hematoxylin-eosin stain.

View larger version (117K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 6. AA/AG Barrett's esophagus high-grade dysplasia. (A) CD1 protein expression by immunohistochemistry; (B) hematoxylin-eosin stain.

View larger version (28K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 7. Chromosome instability in Barrett's esophagus (BE). (A) Chromosome polysomy index in BE metaplasia, low-grade dysplasia, high-grade dysplasia. Chromosome 9 FISH in BE high-grade dysplasia, (B) polysomy (arrows), and (C) diploidy (arrows). (D) Cyclin D1 expression and chromosome polysomy in BE. (*) Indicates statistical significance. AA/AG genotype; A, adenosine; G, guanine; Met, metaplasia; LGD, low-grade dysplasia; HGD, high-grade dysplasia; CPI, chromosome polysomy index; CD1, cyclin D1; WMI, weighted mean index.

	DISCUSSION

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The G/A870 polymorphism has been associated with preferential expression of a truncated CD1 protein, which lacks the critical elements necessary for the nuclear to cytoplasmic export and consequently has a sustained nuclear expression.^22,28-30 Through the enhanced nuclear half-life, the alternate CD1 form maintains high levels of protein expression and facilitates cellular transformation.³² The lack of a good quality specific antibody discriminating the two CD1 protein forms represents an obstacle for a precise assessment of CD1 truncated form protein levels. However, in EAC cell lines, both alternate transcript and its encoded protein are expressed in a allele-dose dependent manner³² (and J.G.I, unpublished observation) underscoring our hypothesis that the CD1 G/A870 polymorphism would be associated with increased CD1 alternate form protein levels.

The results of this study provide a mechanistic explanation of the role of CD1 polymorphism in defining clinical biology EAC. Chromosome instability, a form of genomic instability, is a critical driving force for carcinogenesis since it can determine the rate of accumulation of genetic changes.^2,46,47 It can also influence the number of accumulated aberrations, enhancing clonal diversity and permitting the selection of clonal populations bearing a more aggressive phenotype. In vitro studies suggests that CD1 overexpression may drive chromosome instability through centrosomal and mitotic spindle abnormalities.¹⁷ In this study, we assessed the degree of chromosome instability by measuring chromosome 9 polysomy. In previous studies of biomarkers for risk of developing upper aerodigestive tract cancers, we demonstrated that chromosome polysomy, similar to chromosome aneuploidy,⁴⁵ is an indicator of accumulated genetic damage and ongoing genomic instability.^42,44,47-49 Our data suggest that, by enhancing genomic instability, the CD1 A870 allele increases the rate of acquisition of genetic changes, and enhances clonal genetic diversity during EAC evolution.

Of interest, patients bearing the AA or AG genotype had a significantly younger age of cancer diagnosis, which would potentially confer a better survival after surgery. However, the presence of the A allele appeared to impact overall survival irrespective of age and in allele-dose dependent fashion, underscoring the more aggressive tumor phenotype associated with the G/A870 polymorphism.

Taken together, our data suggests that AA/AG genotype promotes increased levels of CD1 protein, thereby enhancing the acquisition and the extent of genetic changes and leading to a more rapid development of cancer and poor outcome.

Our novel findings need verification in larger studies. It would be important to develop a molecular-based model of prognosis. If successful, it could lead to early attempts at individualization of therapy for patients with EAC. In addition, strategies directed to modulate CD1 expression may be useful in the therapy of patients with EAC.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Julie G. Izzo

Financial support: Walter N. Hittelman, Jaffer A. Ajani

Administrative support: Walter N. Hittelman, Jaffer A. Ajani

Provision of study materials or patients: Tsung-Teh Wu

Collection and assembly of data: Julie G. Izzo, Jennifer Pan

Data analysis and interpretation: Julie G. Izzo, Tsung-Teh Wu, Xifeng Wu, Joe Ensor, Walter N. Hittelman, Jaffer A. Ajani

Manuscript writing: Julie G. Izzo, Xifeng Wu, Rajyalakshmi Luthra, Stephen G. Swisher, Clifford K.S. Chao, Walter N. Hittelman, Jaffer A. Ajani

Final approval of manuscript: Julie G. Izzo, Jaffer A. Ajani

Other: Joe Ensor [Statistical analysis], Arlene Correa [Maintenance of clinical database], Stephen G. Swisher [Treatment of patients]

	ACKNOWLEDGMENTS

 
W.N. Hittelman is a Sophie Caroline Steves professor in cancer research. We thank Ignacio Wistuba, MD, and Lakshmi Kakarala for assistance with tissue preparation.

	NOTES

 
Supported by Grants No. NIH-NCI RO1 DE13157-04 (W.N.H.), NIH-NCI EDRN CA 86390 (M.R.S.), and CA-16672, and The University of Texas M.D. Anderson Multidisciplinary Esophageal Research Grant, Riverkreek Foundation, Dallas, Cantu, Smith, and Park Families (J.A.A.).

W.N.H. and J.A.A. have contributed equally to the performance of the study and preparation of the article.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted June 26, 2006; accepted November 29, 2006.

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