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Journal of Clinical Oncology, Vol 25, No 28 (October 1), 2007: pp. 4358-4364 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.11.2599 Global Histone Modifications Predict Prognosis of Resected Non–Small-Cell Lung Cancer
From the Departments of Medical Oncology and Pathology, Vrije Universiteit Medical Center, Amsterdam, the Netherlands; and Statistics Department, and Department of Thoracic Oncology, Université de la Méditerranée, Assistance Publique Hôpitaux de Marseille, Marseille, France Address reprint requests to Giuseppe Giaccone, MD, PhD, Medical Oncology Branch, CCR, National Cancer Institute, 10 Center Dr, Bldg 10, Room 12N226, Bethesda MD 20892-190; e-mail: giacconeg{at}mail.nih.gov
Purpose Epigenetic modifications may contribute to the development and progression of cancer. We investigated whether epigenetic changes involving multiple histones influence prognosis of non–small-cell lung cancer (NSCLC) patients. Patients and Methods We used immunohistochemistry to assess histone 3 lysine 4 dimethylation (H3K4diMe), and acetylation of histone 2A lysine 5 (H2AK5Ac), histone 2B lysine 12, histone 3 lysine 9 (H3K9Ac), and histone 4 lysine 8 in resected tumor samples of 138 NSCLC patients. Data were analyzed using a recursive partitioning analysis (RPA).
Results The RPA classified the patients into seven distinct prognostic groups based on TNM stage (first node), histology, and histone modifications: H3K4diMe (< or Conclusion The prognostic influence of epigenetic changes involving multiple histones, in particular H2A and H3, is greater in early NSCLC, and evaluation of these changes may help in selecting early-stage NSCLC patients for adjuvant treatment. Our observations provide a rationale for the use of a combination of standard chemotherapy with drugs interacting with histone modifications, such as histone deacetylase inhibitors.
Lung cancer is the leading cause of cancer-related death worldwide, with more than 1.2 million deaths each year.1 Non–small-cell lung cancer (NSCLC) represents approximately 80% of all lung cancers.2 The poor prognosis of NSCLC is due mainly to late diagnosis, inasmuch as only approximately 20% to 30% of patients are eligible for tumor resection.3 Despite surgery, recurrences are common even in early NSCLC, occurring in up to two thirds of patients.4 Although pathologic staging is the most important prognostic factor for patients who undergo resection, there are broad differences within stages,5 which are due to the different biologic behavior of the tumor. Thus, the identification of biologic markers might help to assess more precisely the prognosis and to address more clearly the use of adjuvant therapy. Several markers have been identified that have prognostic implication in resected NSCLC.6,7 However, none is used in routine practice because of conflicting results reported (reviewed by Zhu et al8), technological challenges, or both. In addition to genetic alterations, epigenetic changes may contribute to the development and progression of cancer and leukemia.9 The term epigenetics refers to a number of molecular mechanisms that regulate gene expression without changing the DNA sequence. These mechanisms include alterations in the methylation status of DNA, covalent modification of histone tails, chromatin remodeling, and microRNAs.10-12 Epigenetic deregulation may affect several aspects of tumor cell biology, including cell growth, differentiation, and cell death, and thereby it is likely to be linked to patient prognosis.13 Most studies on the role of epigenetic alterations in cancer have focused on the aberrant pattern of DNA methylation; in fact, the potential targeting of DNA methylation as a therapeutic approach for lung cancer treatment is being evaluated.14 In comparison to DNA methylation, other mechanisms of epigenetic regulation, such as the methylation and acetylation of histones, are less well characterized. However, histone modifications together with DNA methylation seem to have a vital role in organizing nuclear architecture, which in turn is involved in regulating transcription and other nuclear processes (reviewed by Esteller15). A recent report identified the global pattern of histone modification as predictor of the risk of recurrence in prostate cancer patients.16 By applying an unsupervised clustering algorithm, patients were grouped according to the histone H3 and H4 modification patterns determined by immunohistochemistry in a tumor tissue microarray. Using this approach, the authors showed that the groups identified were clinically relevant, and provided prognostic information independently of classical prognostic factors, such as tumor grade, preoperative prostate-specific antigen levels, or capsule invasion. In terms of clinical applicability, patients with a lower risk of recurrence could also be identified by examining a single modification, the dimethylation of histone H3 lysine 4 (H3K4). We investigated the pattern of global histone modification as a potential prognostic marker in resected NSCLC. We used immunohistochemistry to evaluate the level of H3K4 dimethylation (H3K4diMe),16 and the acetylation of four additional histone residues, including histone 2A lysine 5 (H2AK5Ac), histone 2B lysine 12 (H2BK12Ac), histone 3 lysine 9 (H3K9Ac), and histone 4 lysine 8 (H4K8Ac). We analyzed the relationship between epigenetic changes involving multiple histones and patient prognosis using a recursive partitioning analysis (RPA), with overall survival as the primary end point.
Patients Tumor samples of 138 patients with early-stage NSCLC (stage I to IIIA) were included in the present study. Patients underwent radical thoracic surgery of primary tumor (lobectomy or pneumonectomy) and regional lymph nodes at the Vrije Universiteit Medical Center (Amsterdam, the Netherlands), between January 1988 and December 1995. Patients did not receive any preoperative or postoperative treatment. The histopathologic features of the tumor specimens were classified according to the WHO criteria.17 The TNM staging was determined according to the 1997 International Union Against Cancer classification (Table 1). 4 Institutional review board approval was obtained to use archived material for research purposes.
Immunohistochemistry We used specific rabbit polyclonal antibodies to detect five different histone modifications, including H3K4diMe (Abcam, Cambridge, United Kingdom) and H2AK5Ac, H2BK12Ac, H3K9Ac, and H4K8Ac (all from Cell Signaling Technology, Danvers, MA). The use of antibodies was optimized and, once an appropriate dilution and incubation time had been determined, a whole-tissue section of each tumor was stained with each antibody, using standard two-step indirect immunohistochemistry. After deparaffinization in xylenes, the sections were rehydrated in graded alcohols. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol at room temperature (24°C). The sections were placed in a 120°C solution of 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. Primary antibodies were applied as follows: H3K4diMe, 1:1000 for 30 minutes at room temperature; H2AK5Ac, 1:50 overnight; H2BK12Ac, 1:50 overnight; H3K9Ac, 1:100 overnight; H4K8Ac, 1:50 overnight). Detection was accomplished with the Dako (Carpinteria, CA) Envision System, followed by chromogen detection with diaminobenzidine. The sections were counterstained with hematoxylin, dehydrated, and mounted. Negative controls were whole-tissue sections stained in the absence of the primary antibody. A semiquantitative assessment of antibodies staining on whole-tissue sections was performed independently by two observers (F.B. and P.L.), who were blinded to all clinicopathologic variables. In case of discordance, both observers defined the final score of the sample together. The score of each whole-tissue section was based on the frequency, regardless of intensity, of cells with positive nuclear staining (range, 0% to 100%) within the tumor.
Statistical Analysis
An RPA18 was performed to test how histone modifications might influence prognosis. In RPA, a classification tree is constructed that provides decision rules for assigning a sample to a category based on a series of sequential decisions. At each stage, a single predictor is used, and depending on whether the value of the predictor is above or below a selected cutoff value, the sample is assigned to a left or right node. Each of the resulting nodes is then analyzed using the same procedure, although different predictor variables and cutoff values may be used. The cutoff value is selected to maximize the likelihood-ratio
Samples from six patients were not assessable because of insufficient tumor tissue, leaving 132 samples available for analysis. Major clinical and biologic characteristics of these 132 patients are summarized in Table 1.
Immunohistochemical Study of H3K4diMe, H2AK5Ac, H2BK12Ac, H3K9Ac, and H4K8Ac
Global Histone Modification Patterns and Survival The median follow-up was 148 months. One hundred two patients were dead at the time of this analysis, including three lost to follow-up, and 30 were alive, including two patients alive with a local relapse of their disease. The median overall survival and disease-free survival of the whole population was 28 months (95% CI, 13 to 43 months) and 22 months (95% CI, 10 to 33 months), respectively. The RPA classified patients into seven distinct prognostic groups (Fig 2 and Table 2). The first prognostic node was based on pathologic tumor stage, with those with stage II or higher having a worse survival than those with stage I (Fig 2).
Patients with a pathologic tumor stage II or higher were divided into two additional prognostic groups on the basis of their pathologic stage. No other variable could segregate patients with a pathologic tumor stage III. In contrast, pathologic tumor stage II patients were classified into two separate groups by a third node based on acetylation of H2AK5. Patients whose tumors expressed H2AK5Ac in less than 5% of tumor cells had a worse survival compared with patients whose tumors had more than 5% of cells expressing H2AK5Ac (Fig 3).
Within the stage I patients, the RPA also provided a second node based on histology. Stage I patients with an adenocarcinoma had a worse prognosis than those with a large-cell or squamous cell carcinomas. RPA incorporated histone modifications as a third node for stage I patients. Dimethylation of H3K4 significantly influenced survival of stage I patients with large-cell or squamous cell carcinomas, with a greater survival for patients whose tumor expressed high levels (  85% of tumor cells) of H3K4diMe (group 1 v group 2). Moreover, acetylation of H3K9 significantly influenced survival of stage I patients with adenocarcinomas, with a greater survival for patients whose tumors expressed lower levels (< 68% of tumor cells) of H3K9Ac (group 3 v group 4). The seven groups defined the terminal classification of the 132 patients available and were associated with different disease-free (P < .0001) and overall survival (P < .0001; Table 2 and Fig 3). A Cox proportional hazards model including age, sex, pathologic stage, histology, and RPA groups was analyzed to assess independent factors for overall survival. Sex, pathologic stage, and histology were not retained by the model. Conversely, age and RPA groups were found to influence overall survival significantly and independently (Table 3).
In addition to genetic alterations, epigenetic modifications have been implicated in carcinogenesis and cancer development in various types of solid cancer and hematologic malignancies.9-11 Epigenetic modifications seem to have different importance in the development of different tumor types. During prostate carcinogenesis, for example, epigenetic changes are so frequent that they allow discrimination between normal and neoplastic tissue with a specificity of up to 100%.19,20 Furthermore, the prognostic value of certain epigenetic alterations in prostate cancer, breast cancer, and leukemia have been established.13 Conversely, the contribution of epigenetic changes to lung carcinogenesis seems to be less clear.21 In this study, the pattern of expression and biologic relevance of some histone modifications were analyzed for the first time in NSCLC patients who had undergone resection. Relevant features of the histone modification patterns observed in our series include the predominant but not exclusive detection in neoplastic cells in relation to normal lung epithelium, and a characteristic granular staining pattern. The nontumor exclusive staining as well as the low level of heterogeneity noted in some samples is not surprising, giving the role of histone modifications in cell growth, differentiation, and cell death.13 We show that histone H3K4diMe as well as histone H2AK5Ac and H3K9Ac influenced overall and disease-free survival of NSCLC patients who had undergone resection, suggesting that the role of epigenetic modifications in lung carcinogenesis should be explored further. The postoperative management of stage I NSCLC is still a matter of debate. Although surgery alone represents the standard of care for stage IA patients, conflicting results have been presented regarding the use of adjuvant chemotherapy for stage IB patients.22,23 Despite the relatively early diagnosis, a substantial percentage of stage IB patients will experience relapse and succumb to their disease; 5-year survival of pathologic stage IB treated by surgery alone is 57%.4 Stage I represents a heterogeneous group as demonstrated by Potti et al.5 On the basis of a gene-expression profile termed lung metagene, these authors were able to predict recurrence for individual stage I patients significantly better than did clinical prognostic factors, and they identified a subgroup of stage IA patients with a high risk of recurrence. Whether these data provide sufficiently robust and reproducible information regarding the use of adjuvant chemotherapy in early-stage NSCLC remains to be established. On the basis of our results, histone modifications, especially methylation and acetylation of histone H3, should also be considered in the attempt to better define prognostic subgroups of stage I NSCLC patients. Interestingly, several drugs interacting with histone modifications have been developed in recent years, such as the histone deacetylase inhibitors.24 Several structurally distinct classes of histone deacetylase inhibitors have been developed25 and are being tested in clinical trials. A significant antitumor activity was reported in several tumor types, including NSCLC, for some of these agents, such as pivaloyloxymethyl butyrate and CI-994.26,27 Numerous additional studies are still ongoing and results are awaited. In our study we used the tool of RPA to assess the prognostic value of several histone modifications in relation to known prognostic features in resected NSCLC. There are several methodologies to investigate the potential prognostic value of clinical and biologic features. Multidimensional scaling, as used by Seligson et al,16 is a set of data analysis techniques that displays the structure of distance-like data as a geometric picture, on which multiple subsequent analyses usually are performed.28 Conversely, the central result of RPA is a tree, in which the data are organized (partitioned) into nodes (leaves) along branches; data that are more similar according to specified criteria (eg, overall survival) tend to be localized into the same nodes, whereas more dissimilar data tend to occupy different nodes.29 Therefore, no additional analysis is needed. Furthermore, RPA is particularly well suited when the connection between the variables is complex, and even when there is missing information.30 For these reasons we used RPA in this study instead of multidimensional scaling. The selection of pTNM stage, a well-known prognostic factor in NSCLC, as the first node supports the validity of the method. Therefore, the histone modifications retained by the RPA should be regarded as valuable new prognostic factors in resected NSCLC patients; in particular, histone H3K4diMe and H3K9Ac, which are related to prognosis of those patients with resected stage I NSCLC. However, how these specific epigenetic modifications or combination of modifications affect the expression of particular genes or tumor behavior remains to be fully understood. One possibility is that expression of critical genes may be modulated differently by different epigenetic changes, and this hypothesis should be evaluated further by comparative transcriptional analysis of tumors characterized by different patterns of histone modifications. In conclusion, our results highlight the role of epigenetic changes involving multiple histones in lung cancer, identifying possible useful markers for selection of early-stage (especially stage I) NSCLC patients for adjuvant therapy. Our observations provide a rationale for studies of standard chemotherapy, drugs interacting with histone modifications, or combinations of both for the management of these patients.
The author(s) indicated no potential conflicts of interest.
Conception and design: Giuseppe Giaccone, Anderson Loundou, Simone W. Span, Frank A.E. Kruyt, Jose Antonio Rodriguez Financial support: Giuseppe Giaccone Administrative support: Giuseppe Giaccone, Jose Antonio Rodriguez Provision of study materials or patients: Giuseppe Giaccone, Simone W. Span, Jose Antonio Rodriguez Collection and assembly of data: Fabrice Barlési, Giuseppe Giaccone, Marielle I. Gellegos-Ruiz, Simone W. Span, Pierre Lefesvre, Frank A.E. Kruyt, Jose Antonio Rodriguez Data analysis and interpretation: Fabrice Barlési, Giuseppe Giaccone, Anderson Loundou, Frank A.E. Kruyt, Jose Antonio Rodriguez Manuscript writing: Fabrice Barlési, Giuseppe Giaccone, Anderson Loundou, Frank A.E. Kruyt, Jose Antonio Rodriguez Final approval of manuscript: Fabrice Barlési, Giuseppe Giaccone, Marielle I. Gellegos-Ruiz, Anderson Loundou, Simone W. Span, Pierre Lefesvre, Frank A.E. Kruyt, Jose Antonio Rodriguez
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test or Fisher's exact test was used according to test condition. A Cox proportional hazards model was performed to establish independent factor(s) for survival. Statistical significance was defined as P < .05. All of the tests are two sided. Statistical analysis was performed using the SPSS software package, version 13.0 (SPSS Inc, Chicago, IL). 
