Imaging, Diagnosis, Prognosis

Decreased Expression of Cluster at 1q21 Defines Molecular Subgroups of Chemoradiotherapy Response in Esophageal Madan G. Luthra,1JafferA. Ajani,2 Julie Izzo,3 Joe Ensor,4 Ts un g-Teh Wu, 1Asif Rashid,1Li Zhang,4 Alexandria Phan,2 Norio Fukami,5 and Rajyalakshmi Luthra6

Abstract Purpose: Resistance to preoperative chemoradiotherapy (CTXRT) in 75% of patients with esophageal adenocarcinoma (EAC) underscores the need for identification of biomarkers of CTXRT response. We previously noted an association between decreased expression of epidermal differentiation complex (EDC) S10 0A2 and SPRR3 at chromosome 1q21 and CTXRT resistance. In the current study, we did an in-depth investigation of the expression of 1q21-1q25region genes to uncover the role of the EDC and its flanking genes in CTXRTresponse. Experimental Design: We compared 19 pretreatment EAC specimens with normal squamous mucosa for the expression of 517 genes at chromosome 1q21-1q25 and selected target genes based on their differential expression. Using the pathologic complete-response (pathCR) status of the resected specimens as a representation of CTXRTsensitivity, we assessed the association between the expression of target genes and CTXRTresponse and clinical outcomes. Results: On the basis of the expression levels of IVL, CRNN, NICE-1, S100A2,andSPPR3,genes within and in close proximity to the EDC, cancers were segregated into high (subgroup I) or low (subgroup II) expressers. Four of the five pathCRs were high expressers. Thus, low expressers, with one exception, were all nonresponders. Patients in subgroup I also had longer survival than those in subgroup II, although this result was not statistically significant owing to the small study number. Conclusions:The expressionlevels of genes mapping within and close to the EDC define CTXRT response subgroups in EACs.

Esophageal remains one of the most fatal malignancies, chronic reflux esophagitis (4). When EAC is local-regional, with a 5-year survival rate of <20%. Over the past two decades, preoperative chemoradiation (CTXRT) is commonly recom- a remarkable shift has occurred in the epidemiology of eso- mended. However, the role of preoperative CTXRT remains phageal cancer, resulting in an alarming increase in the inci- controversial. When CTXRT is used, longer survival is noted in a dence of adenocarcinoma of the proximal esophagus, with a small fraction (about 27%) of patients who achieve pathologic relative decline in the incidence of esophageal squamous cell complete response (pathCR; refs. 5–9); however, patients who carcinoma (1). The incidence of esophageal adenocarcinoma are likely to have a pathCR cannot be predicted by the (EAC) is increasing faster than that of any other type of cancer, pretreatment clinical parameters. Thus, it is of paramount at a yearly rate of f10%, and EAC ranks in the top 15 cancers importance to discover biomarkers that can predict response to among Caucasian men in the United States (1–3). CTXRT to individualize and optimize therapy for this group of Most EACs arise in the background of Barrett’s esophagus, patients. We believe that individualization of therapy may be which is characterized by the replacement of normal squamous possible by studying the molecular biology of EAC and the epithelium with metaplastic columnar epithelium due to patient’s genetics. In an expression profiling pilot study, designed to identify molecular signatures predictive of response to therapy, we S100A2 Authors’ Affiliations: Departments of 1Pathology, 2Gastrointestinal Medical found substantially lower expression of the (S100 3 4 SPRR3 Oncology, Experimental Therapeutics, Biostatistics and Applied Math, calcium-binding A2) and (small proline-rich 5Gastrointestinal Medicine and Nutrition, and 6Hematopathology,The University of protein 3 or esophagin) genes in pretreatment cancer speci- Texas M. D. Anderson Cancer Center, Houston,Texas mens of patients resistant to CTXRT (10). The loss of expression Received 6/28/06; revised 11/7/06; accepted 11/10/06. of both S100A2 and SPRR3 has been associated with 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 premalignant and malignant states, including cancers of the with 18 U.S.C. Section 1734 solely to indicate this fact. lung, esophagus, and cervix (11–18). Both genes are located in Requests for reprints: Rajyalakshmi Luthra, Division of Pathology and an evolutionarily conserved genetic cluster, designated as the Laboratory Medicine, University of Texas M. D. Anderson Cancer Center, 8515 epidermal differentiation complex (EDC), at the 1q21 chro- Fannin St., NAO1.091, Houston, TX 77054. Phone: 713-794-5443; Fax: 713-794- 4773; E-mail: [email protected]. mosomal band (19–21). This genetic region encompassing F 2007 American Association for Cancer Research. 2 Mbof genomic DNA harbors3 gene families and appro- doi:10.1158/1078-0432.CCR-06-1577 ximately 43 genes that are involved in terminal squamous

Clin Cancer Res 2007;13(3) February1,2007 912 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Differentiation Genes in Esophageal Cancer Resistance differentiation of the epidermis (19). A recent study has no contraindications for surgery. If a patient had an R0 resection, suggested that the expressional down-regulation of some EDC no further therapy was planned. Patients who underwent an R1 complex genes, including S100A2 and SPRR3, may serve as a resection (microscopic carcinoma at the margin) or R2 resection potential marker of progression to EAC (22). (gross carcinoma after surgery) or who had M1 disease were offered palliative care. We hypothesized that the EDC gene cluster is differentially Each patient was assessed at 3, 6, 9, and 12 months after surgery and expressed in CTXRT-sensitive and CTXRT-resistant EAC, and then every 6 months for 2 additional years and then every year or until that this may reflect distinct biological EAC entities. To test this death. Local-regional recurrence was defined as recurrence within the hypothesis, we compared pretreatment specimens of EAC and surgical field or mediastinal nodes. Metastatic cancer was defined as normal squamous esophageal mucosa for the expression of evidence of cancer outside the regional area, such as in the bone, brain, approximately 517 genes in the 1q21-1q25 chromosomal liver, or lung. region that included the EDC cluster and its flanking regions Tissue specimens and collection. All tissue specimens were obtained and selected target genes based on their maximal differential during a diagnostic preoperative endoscopic procedure through a expression. Then, using pathCR after therapy as an end point, protocol approved by the M. D. Anderson Cancer Center Institutional we analyzed the association between the expression levels of Review Board and after informed consent was obtained from patients. Both normal squamous mucosal (NSM) tissue and cancer tissue were 1q21-1q25 target genes and response to CTXRT and clinical collected from each patient. The size of a typical biopsy specimen was outcome. f2.0 mm. Biopsy specimens were placed in cryogenic vials, snap frozen in liquid nitrogen, and stored at À80jC until further use. In this Materials and Methods report, pretreatment cancers from 19 patients and NSM tissue from 7 of these patients with high-quality RNA were analyzed. Patient selection and evaluation. Nineteen patients, including 16 Histologic evaluation. For each specimen analyzed by microarray, from a previous report (10), with localized, histologically confirmed an adjacent tissue biopsy was given to a pathologist (TTW or AR) to adenocarcinoma of the thoracic esophagus were included in the study. confirm the presence of cancer and its histology. Routine H&E-stained All 19 patients participated in a clinical trial approved by The slides were used to evaluate the presence of cancer in pretreatment University of Texas M. D. Anderson Cancer Center’s Institutional endoscopic biopsies and esophagectomy specimens. The pathologic Review Board. Patients with tumors classified as T2-3 with any N, response in the resected esophagus was assigned to one of two patients with M1a cancer (celiac nodes associated with a gastroesoph- categories: no residual carcinoma in the esophagus (pathCR) or the ageal junction carcinoma), and patients with T1N1 carcinoma were presence of any cancer cells in the resected specimen (

Table 1. Patient and cancer characteristics

Specimen Gender Differentiation Barrett’s association % tumor cells in resected specimen

Malignant subgroup I 1 F Moderate No 0 3 M Moderate No 0 4 M Moderate to poor Yes No surgery 6 M Moderate No 1-10 12 M Poor No 1-10 16 M Well to moderate No 0 19 F Moderate No 11-50 22 M Poor No No surgery 23 M Poor Yes 0 Malignant subgroup II 8 M Moderate Yes 1-10 11 M Poor Yes 1-10 13 M Moderate No 0 14 M Moderate Yes 1-10 18 M Poor Yes No surgery 20 M Moderate No 11-50 25/44 M Poor No 1-10 26 M Poor No (>50) 28/38 M Moderate No (>50) 29/36 M Poor Yes (>50)

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Fig. 1. Treeview displays of unsupervised cluster analysis of esophageal cancers and normal squamous mucosa (NSM). Expression data of 517 genes from the chromosome 1q21-1q25region were used for cluster analysis as described in Methods. All NSMs clustered together and segregated from cancers (A and B). However, cancers segregated into two clusters designated as subgroups I and II (A and B). Four of the five cancers that achieved pathologic complete response (pathCR; red stars), clusteredinsubgroupI.C, genes with marked difference in expression between the subgroups.

The Affymetrix U133A GeneChip contains 22,215 probe sets that insufficient RNA in five cancers (numbers 6, 20, 22, 23, and 38), we correspond to 14,593 well-characterized human genes.7 Microarray were able to evaluate only 14 cancers by real-time qPCR. For cancer Suite 5.0 software and custom tools developed by the M. D. Anderson specimens, triplicates of 100 ng of total RNA were reverse transcribed in Bioinformatics Department were used to analyze the data. The a final volume of 20 AL using random primers and SuperScript II microarray data were processed using the position-dependent nearest Reverse Transcriptase (Invitrogen, Carlsbad, CA). The final reaction neighbor model to normalize and extract values (23). products from triplicates were pooled and stored at À20jC until further The genes with below-median expression value were regarded as absent use. For normal specimens, equal amounts of RNA from each of the genes. The expression data of probe sets present in the 1q21-1q25 seven NSM biopsies were pooled first and then 100 ng of the pooled region representing 517 genes were extracted and processed using an RNA was reverse transcribed in triplicate as described above. For both unsupervised hierarchical clustering algorithm (24). The cluster analysis cancer and normal samples, total RNA from the same aliquot of RNA was done using the uncentered Pearson correlation as similarity metric that was used for microarray analysis was used for reverse transcription. and average linkage algorithm to combine cluster branches. Differen- The TaqMan minor grove binder probe and the ABI Prism 7900 HT tially expressed genes were identified using the standard t test. Sequence Detection System (PE Applied Biosystems, Foster City, CA) Real-time quantitative PCR. We did the real-time quantitative PCR were used for detecting real-time PCR products. Primers and probes for (qPCR) of nine genes that had average expression differences of 2-fold the target and internal control gene (18S) were designed by PE Applied or greater between the NSM and cancer specimens in microarray Biosystems and obtained via their Assays-on-Demand Gene expression analysis. The genes were RGS5 (regulator of G-protein signaling 5), products services. PCR assays included 10 AL of TaqMan Universal ADAR (adenosine deaminase, RNA specific), ECM1 (extracellular Master Mix No Amperase UNG (2Â), 1 ALof20Â Assays-on-Demand matrix protein 1), IVL (involucrin), CRNN (cornulin), NICE-1, SPPR3, Gene Expression Assay Mix, and 2 AL of cDNA diluted in Rnase-free S100A2, and CRABP2 (cellular retinoic acid-binding protein 2). Due to water, in a final volume of 20 AL. The PCR thermal cycling conditions were as follows: 10 min at 95jC for AmpliTaq Gold activation and 40 cycles for the melting (95jC, 15 s) and annealing/extension (60jC, 7 Available online: http://www.affymetrix.com/support/technical/libraryfilesmain.affx 1 min) steps. Each target was amplified individually and in duplicate.

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ÀDC T Comparative CT method (2 ) for relative quantification of gene expression. The expression of each target was calculated based on the Table 2. Genes differentially expressed in difference between amplification of the individual target mRNA esophageal cancers template and the internal control (18S) mRNA template using the Gene symbol Gene name DCT method. The relative expression levels of target genes in comparison to the control gene were then calculated using the formula ÀD Down-regulated in cancers CT D * 2 , where CT represents the difference between each target gene CRNN Cornulin ( open and the control gene (average CT for the target minus average CT for 18S reading frame 10, C1orf10) ÀD RNA). To simplify the data presentation, we multiplied 2 CT values by NICE-1 * Chromosome 1 open reading a factor of 1,000,000. frame 42, C1orf42 SPRR3 Statistical methods. The relative expression values obtained by real- Small proline-rich protein 3 ECM1 * Extracellular matrix protein 1 time qPCR were used to determine the potential of the genes to S100A9 IVL, S100 calcium-binding protein A9 discriminate pathCR from

www.aacrjournals.org 915 Clin Cancer Res 2007;13(3) February 1,2007 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis in cancer and NSM specimens (P < 0.01; t test). Table 2 shows S100A2, the three genes within the EDC (Fig. 3B). Among the the list of genes differentially expressed in the cancer specimens. genes within 0.5 Mbof the EDC cluster, the levels of CRNN and Both cancer subgroups showed higher expression (z2-fold) of NICE-1 were also substantially lower in subgroup II compared 18 genes compared with NSM. Lower expression (z2-fold) of with subgroup I. The levels of ECM1 and CRABP2, two genes 35 genes was seen in cancer versus NSM specimens (Fig. 2). The flanking either side of the EDC (about 3 Mb apart), were, expression levels of several of these genes differed substantially however, similar in both groups. A clear segregation of the two among cancer subgroups. For instance, 14 genes (40%) showed subgroups, without any overlap in the expression levels, was a greater than 4-fold decrease in subgroup II cancers, whereas seen in CRNN and NICE-1. The dramatic differences in the only 5 of the 14 genes showed such low expression in subgroup expression levels of CRNN, NICE-1, IVL, SPRR3, and S100A2 I cancers (Table 2). segregated the cancers into high (subgroup I) and low Intriguingly, within the cancer subgroups, the expression expressers (subgroup II). It should be noted that substantial pattern suggested a gradient of decreased expression culminat- heterogeneity was found in gene expression levels among the ing with the maximal down-regulation of genes included in the cancers in both subgroups. EDC cluster at 1q21. Thus, we focused our analysis on a 5-Mb It has been suggested that within the 1q21 region, the EDC DNA region located on 1q21-1q23 and encompassing the EDC gene cluster and its closely flanking genes may have a genes. A schematic of the chromosomal region, with a detailed coordinated transcription control mechanism (25–27). Hence, map of the EDC cluster and flanking genes, is shown in Fig. 3A. using the data obtained by real-time qPCR, we analyzed the Using real-time qPCR, we analyzed the level of transcriptional correlation between the expression levels of 1q21 target genes expression of (a) three genes included in the EDC (IVL and using linear relationship analysis methods. Strikingly, as shown SPPR3, both members of the cornified envelop precursor in Table 3, we found the presence of statistically significant family, and S100A2) and (b) six genes flanking the EDC region coordinated down-regulation of the EDC genes and their (ECM1, ADAR, RGS5, CRNN, NICE-1, and CRABP2). neighboring genes, CRNN, NICE-1, and CRABP2. Real-time qPCR data confirmed the results obtained by Molecular malignant subgroups, clinical characteristics, and microarray analysis. Compared with normal squamous epithe- patient outcome. The 1q21-1q25 molecular malignant sub- lium, the expression levels of ECM1, IVL, CRNN, NICE-1, groups were not associated with patient characteristics, includ- SPRR3, S100A2, and CRABP2 were lower in cancers, whereas ing age at diagnosis, clinical stage, and location of the primary the levels of ADAR and RGS5 were slightly higher in cancers. tumor. It is noteworthy that five (71%) of the seven patients Among cancer subgroups, subgroup II had substantially with EAC that developed in the background of Barrett’s lower levels than subgroup I of expression of IVL, SPRR3, and esophagus were in subgroup II (Table 1).

Fig. 2. Average expression levels of genes differentially expressed in normal and cancer specimens. Graph highlights the dramatic drop in transcription of genes within and in close proximity to the epidermal differentiation complex cluster in esophageal cancers, compared with NSM, and divergence of the two molecular subgroups based on the expression levels of these genes. All genes showing greater than or equal to 2-fold difference in expression were plotted in the order that matched their location on the chromosome, from centromere to telomere.

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Fig. 3. Real-time qPCR expression levels of nine genes: ECM1, CRNN, NICE-1, INV, SPRR3, S100A2, ADAR, CRABP2 and RAG5. A, schematic of the chromosomal region with detailed map of EDC and flanking genes. Bold, genes analyzed by qPCR (B). Relative expression values of the selected genes shown in (B)were calculated as described in Methods using ÀD the 2 CT method. Solid black, relative values for pooled NSM. Dotted pattern and solid gray, relative levels of cancers from subgroups I and II, respectively.The numbers shown in the legend key correspond to patient numbers inTable 1. Dashed lines, expression levels used for dichotomization into high/low expression (high expression was defined as relative expression levels of >100 for CRNN and >5000 for NICE-1 and IVL).

The molecular malignant subgroups seemed to be associated expression levels of genes within the EDC, it was not possible to with response to CTXRT. Four (80%) of the five patients segregate responders from nonresponders within subtypes. achieving pathCR were clustered in molecular subgroup I com- Patients in molecular subgroup II had worse OS, with a pared with only one patient (20%) in subgroup II, suggesting median time of 34 months (95% CI, 16-52 months) compared that subgroup II cancers were resistant to CTXRT. Although with 44 months (95% CI, 0-90 months) for those in subgroup expression levels of CRNN, NICE-1, IVL, SPRR3,andS100A2 I; however, owing to the small cohort size, no statistically were substantially different in the two EAC subgroups (Fig. 3), significant difference was reached (P = 0.55; log-rank test). At a statistically significant relationship between these biomarkers 3 years, 32% of subgroup II survived compared with 78% of and pathCR was not shown by univariate logistic regression subgroup I. Similarly, molecular subgroup II was associated analysis. Three biomarkers, IVL, NICE-1,andCRNN,onthe with shorter DFS, with a median DFS time of 14 months (95% basis of minimal overlap in the expression levels between the CI, 2-26 months) compared with 37 months (95% CI, 0-82 two subgroups of cancers, were dichotomized into high/low months) for molecular subgroup I (P = 0.20; log-rank test). At expression (high expression was defined as relative expression 3 years, 44% of patients in subgroup II were disease-free levels of >100 for CRNN and >5,000 for NICE-1 and IVL). compared with 78% in subgroup I. Then, a two-sided Fisher’s exact test was used to investigate the association of the dichotomized biomarkers with pathCR. A Discussion statistically significant association with pathCR was observed only for IVL (P = 0.05). However, the small sample size may Chemoradiation resistance and local recurrence or distant have underpowered the study. Due to the heterogeneity in the metastases are clinical features of over 75% of esophageal

www.aacrjournals.org 917 Clin Cancer Res 2007;13(3) February 1,2007 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis cancers treated with CTXRT. Thus, understanding the biological et al. (22), imply that the down-regulation of genes in this properties that render a cancer sensitive or insensitive to chromosomal region is involved in EAC development and chemoradiation is crucial for tumor management. progression. Our report also shows an association between To begin to understand biomarkers predictive of response to these genes and CTXRT response. CTXRT, we previously conducted a transcriptional profiling Recognizing that our data are preliminary, we can still study of pretreatment esophageal cancer biopsies derived from ponder some key observations stemming from the current patients treated on a preoperative CTXRT protocol. Among the study. These include (a) the degree of transcriptional suppres- markers associated with the lack of response to CTXRT, we sion of genes mapping within and close to the EDC can observed a down-regulation of genes located at the chromo- segregate EAC into low and high expressers; (b) the high somal region 1q21. The present analysis of the 517 genes expressers, while showing transcriptional suppression in the within the 1q21-1q25 region clearly shows EAC resistant to EDC cluster region, seem to have a transcriptional signature CTXRT clustered together with a homogenous down-regulation closer to that of NSM; (c) the high expressers are more likely to of genes in this region compared with EAC sensitive to CTXRT. be sensitive to CTXRT; and (d) the majority of the cancers with The in-depth analysis of the genes included in the 1q21-1q25 associated Barrett’s metaplasia cluster in CTXRT-resistant cancer region suggests a gradient of expression changes between cancer subgroup II. Thus, these observations suggest that EAC may molecular subgroups. EAC sensitive to CTXRT harbored a include biologically and molecularly different entities: sub- transcriptional profile closer to that of NSM, whereas resistant group I maintaining similarity to NSM and subgroup II having EAC showed a dramatically different profile stemming mainly a molecular signature similar to glandular epithelium. Of from the differential expression of the EDC gene cluster, importance, cancers within subgroup I were more sensitive to including CRNN, NICE-1, IVL, SPRR3, and S100A2 (Fig. 3A CTXRT, similar to esophageal squamous cell carcinoma, for and B). Relative expression levels of these genes, as determined which the 3-year survival rate after CTXRT is higher than that by quantitative PCR, clearly segregate the two cancer types. for EAC (30, 31), and cancers within subgroup II were resistant The encoded within the EDC are implicated in the to CTXRT, similar to EAC associated with Barrett’s metaplasia terminal differentiation of keratinocytes, and their expression (32). Our study, albeit small, supports the clinical observation is temporarily physiologically coordinated to mediate cessa- that EAC associated with Barrett’s metaplasia is more resistant tion of proliferation (e.g., S100As) and migration toward the to chemoradiation. Further studies are necessary to determine superficial layers (e.g., IVL), with associated progressive corni- which molecular pathways are important for imparting chemo- fication (e.g., SPRRs). The temporal expression of these radiation resistance in subgroup II. proteins seems to be a biological program, coordinated by Cellular retinoic acid-binding protein II, encoded by the subchromosomal position of gene territories and similar CRABP2, is an intracellular lipid-binding protein that associates to that involving the MHC genes (27). The EDC cluster is also with retinoic acid with a subnanomolar affinity. Studies have critical for the maturation and maintenance of the stratified shown that retinoic acid regulates the expression of markers of squamous normal esophageal mucosa. Although the functions differentiation (33–35). Because it has been reported that the of the proteins encoded by the NICE-1, IVL, SPRR3, and cellular retinoic acid-binding protein II enhances the transcrip- S100A2 genes in terminally differentiating keratinocytes are tional activity of the nuclear receptor, the retinoic acid receptor, well documented, the role of CRNN in the differentiation by delivering retinoic acid to this receptor, the retinoic acid program is poorly understood. CRNN is either dramatically pathway may be affected by decreased expression of CRABP2 in reduced or absent in primary esophageal cancer tissues, sug- these cancers. gesting that it is esophageal specific and cancer related Our results, along with those of other investigators, provide (28, 29). Our data, along with a previous report by Kimchi independent confirmation that EDC genes are differentially

Table 3. Linear correlation analysis of log expression levels showing coordinate regulation of target genes

Gene name SPRR3 S100A2 CRNN NICE-1 CRABP2 ECM1 IVL

SPRR3 1.00000 0.31056 0.75039 0.57437 0.49195 À0.41611 0.35021 0.3017 0.0031 0.0401 0.0877 0.1573 0.2408 S100A2 0.31056 1.00000 0.67695 0.58679 0.51717 0.07853 0.40646 0.3017 0.0110 0.0350 0.0703 0.7987 0.1681 CRNN 0.75039 0.67695 1.00000 0.82184 0.57673 À0.04184 0.51497 0.0031 0.0110 0.0006 0.0391 0.8920 0.0717 NICE-1 0.57437 0.58679 0.82184 1.00000 0.69831 0.08612 0.61769 0.0401 0.0350 0.0006 0.0079 0.7797 0.0245 CRABP2 0.49195 0.51717 0.57673 0.69831 1.00000 À0.03677 0.80557 0.0877 0.0703 0.0391 0.0079 0.9051 0.0009 ECM1 À0.41611 0.07853 À0.04184 0.08612 À0.03677 1.00000 À0.04950 0.1573 0.7987 0.8920 0.7797 0.9051 0.8724 IVL 0.35021 0.40646 0.51497 0.61769 0.80557 À0.04950 1.00000 0.24080.1681 0.0717 0.0245 0.0009 0.8724

NOTE: Statistically significant correlation is seen between CRNN and NICE-1, S100A2, SPRR3, CRABP2 ,andIVL and between CRABP2 and CRNN, NICE-1 ,andIVL, as indicated by P values shown in bold.

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regulated in EAC and NSM and validate these genes as markers in addition to previously shown SPRR3 and S100A2, as mar- of EAC. To our knowledge, however, our study is the first to kers of resistance to CTXRT in EAC. report coordinated down-regulation of keratinocyte differenti- ation genes and CRABP2 and the association between trans- Acknowledgments criptional suppression of differentiation-associated genes and We thank Enrique Lopez-Alvarez and Jaime Bailey for their technical assistance, resistance to chemoradiation in EAC. Studies using a larger Dawn Chalaire for critical editing of the manuscript, and James Gilbert for his assis- sample set are in progress to validate IVL, CRNN, and NICE-1, tance in preparing the illustrations.

References 1. Pohl H, Welch HG. The role of overdiagnosis and expression is associated with cell growth arrest. Arch 24. Eisen MB, Spellman PT, Brown PO, Botstein D. reclassification in the marked increase of esophageal Histol Cytol 1998;61:163 ^ 78. Cluster analysis and display of genome-wide expres- adenocarcinoma incidence. J Natl Cancer Inst 2005; 13. Hibi K, Fujitake S, TakaseT, et al. Identification of sion patterns. Proc Natl Acad Sci U S A 1998;95: 97:142 ^ 6. S10 0A2 as a target of the DNp63 oncogenic pathway. 14 8 6 3 ^ 8. 2. Pera M. Recent changes in the epidemiology of Clin Cancer Res 2003;9:4282 ^ 5. 25. Zhao XP, Elder JT. Positional cloning of novel skin- esophageal cancer. Surg Oncol 2001;10:81 ^ 90. 14. Chen BS,Wang MR, Cai Y, et al. Decreased expres- specific genes from the human epidermal differentia- 3. Pera M. Trends in incidence and prevalence of spe- sion of SPRR3 in Chinese human esophageal cancer. tion complex. Genomics 1997;45:250 ^ 8. cialized intestinal metaplasia, Barrett’s esophagus, Carcinogenesis 2000;21:2147 ^ 50. 26. Mischke D.The complexity of gene families involved and adenocarcinoma of the gastroesophageal junc- 15. Smolinski KN, Abraham JM, Souza RF, et al. Acti- in epithelial differentiation. Keratin genes and the epi- tion. World J Surg 2003;27:999 ^ 1008; discussion vation of the esophagin promoter during esophageal dermal differentiation complex. Subcell Biochem 6^8. epithelial cell differentiation. Genomics 2002;79: 19 9 8;31:71 ^ 10 4. 4. Spechler SJ. Clinical practice. Barrett’s esophagus. 875 ^ 80. 27. Williams RR, Broad S, Sheer D, Ragoussis J. Sub- N Engl J Med 2002;346:836 ^ 42. 16. Kimos MC, Wang S, Borkowski A, et al. Esophagin chromosomal positioning of the epidermal differentia- 5. Chirieac LR, Swisher SG, Ajani JA, et al. Posttherapy and proliferating cell nuclear (PCNA) are bio- tion complex (EDC) in keratinocyte and lymphoblast pathologic stage predicts survival in patients with markers of human esophageal neoplastic progression. interphase nuclei. Exp Cell Res 2002;272:163 ^ 75. esophageal carcinoma receiving preoperative chemo- Int J Cancer 2004;111:415^ 7. 28. Contzler R, Favre B, Huber M, Hohl D. Cornulin, a radiation. Cancer 2005;103:1347 ^ 55. 17. Suzuki F, Oridate N, Homma A, et al. S100A2 new member of the ‘‘fused gene’’ family, is expressed 6. Rohatgi P,Swisher SG, Correa AM, et al.Characteriza- expression as a predictive marker for late cervical during epidermal differentiation. J Invest Dermatol tionofpathologic complete responseafterpreoperative in stage I and II invasive squamous cell 2005;124:990 ^ 7. chemoradiotherapy in carcinoma of the esophagus carcinoma of the oral cavity. Oncol Rep 2005;14: 29. Xu Z, Wang MR, Xu X, et al. Novel human esopha- and outcome after pathologic complete response. 14 93 ^ 8. gus-specific gene c1orf10: cDNA cloning, gene struc- Cancer 2005;104:2365^72. 18. Shimada A, Kano J, IshiyamaT, et al. Establishment ture, and frequent loss of expression in esophageal 7. Berger AC, Farma J, Scott WJ, et al. Complete of an immortalized cell line from a precancerous lesion cancer. Genomics 2000;69:322^ 30. response to neoadjuvant chemoradiotherapy in eso- of lung adenocarcinoma, and genes highly expressed 30. Naughton P, Walsh TN. Pre-operative chemo- phageal carcinoma is associated with significantly im- in the early stages of lung adenocarcinoma develop- radiotherapy improves 3-year survival in people with proved survival. J Clin Oncol 2005;23:4330 ^ 7. ment. Cancer Sci 2005;96:668 ^ 75. resectable esophageal cancer. Cancer Treat Rev 8. Darnton SJ, Archer VR, Stocken DD, Mulholland PJ, 19. Volz A, Korge BP, Compton JG, Ziegler A, Steinert 2004;30:141 ^ 4. Casson AG, Ferry DR. Preoperative mitomycin, ifosfa- PM, Mischke D. Physical mapping of a functional clus- 31. Varadhachary GR, Hoff PM. Front-line therapy for mide, and cisplatin followed by esophagectomy in ter of epidermal differentiation genes on chromosome advanced colorectal cancer: emphasis on chemother- squamous cell carcinoma of the esophagus: patho- 1q21. Genomics 1993;18:92^9. apy. Semin Oncol 2005;32:S40 ^ 2. logic complete response induced by chemotherapy 20. Mischke D, Korge BP, Marenholz I,Volz A, ZieglerA. 32. Agarwal B, Swisher SG, Ajani J, et al. Differential leads to long-term survival. J Clin Oncol 2003;21: Genes encoding structural proteins of epidermal corni- response to preoperative chemoradiation and surgery 4009^15. fication and S100 calcium-binding proteins form a in esophageal adenocarcinomas based on presence of 9. Rohatgi P, Swisher SG, Correa AM, et al. Failure gene complex (‘‘epidermal differentiation complex’’) Barrett’s esophagus and symptomatic gastroesopha- patterns correlate with the proportion of residual on human chromosome 1q21. J Invest Dermatol 1996; geal reflux. AnnThorac Surg 2005;79:1716 ^ 23. carcinoma after preoperative chemoradiotherapy for 106:989^92. 33. Hohl D, de Viragh PA, Amiguet-Barras F, Gibbs S, carcinoma of the esophagus. Cancer 2005;104: 21. Steinert PM, Candi E, Kartasova T, Marekov L. Small Backendorf C, Huber M. The small proline-rich pro- 1349^55. proline-rich proteins are cross-bridging proteins in the teins constitute a multigene family of differentially reg- 10. Luthra R, WuTT, Luthra MG, et al. Gene expression cornified cell envelopes of stratified squamous epithe- ulated cornified cell envelope precursor proteins. profiling of localized esophageal carcinomas: associa- lia. J Struct Biol 1998;122:76 ^ 85. J Invest Dermatol 1995;104:902^ 9. tion with pathologic response to preoperative chemo- 22. Kimchi ET, Posner MC, ParkJO, et al. Progression of 34. Asselineau D, Bernard BA, Bailly C, Darmon M. radiation. J Clin Oncol 2006;24:259 ^ 67. Barrett’s metaplasia to adenocarcinoma is associated Retinoic acid improves epidermal morphogenesis. 11. Nagy N, Brenner C, Markadieu N, et al. S100A2, a with the suppression of the transcriptional programs Dev Biol1989;133:322^35. putative tumor suppressor gene, regulates in vitro of epidermal differentiation. Cancer Res 2005;65: 35. Hohl D, Lichti U, Breitkreutz D, Steinert PM, Roop squamous cell carcinoma migration. Lab Invest 2001; 3146 ^ 54. DR. Transcription of the human loricrin gene in vitro 81:599^612. 23. Zhang L, Miles MF, Aldape KD. A model of molecu- is induced by calcium and cell density and sup- 12. Hitomi J, Kimura T, Kusumi E, et al. Novel S100 lar interactions on short oligonucleotide microarrays. pressed by retinoic acid. J Invest Dermatol 1991;96: proteins in human esophageal epithelial cells: CAAF1 Nat Biotechnol 2003;21:818 ^ 21. 414 ^ 8.

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Madan G. Luthra, Jaffer A. Ajani, Julie Izzo, et al.

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