Originally published as JCO Early Release 10.1200/JCO.2005.02.998 on April 25 2005

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Protein Expression of a Triad of Frequently Methylated Genes, p73, p57^Kip2, and p15, Has Prognostic Value in Adult Acute Lymphocytic Leukemia Independently of Its Methylation Status

Carlos Bueso-Ramos, Yunling Xu, Timothy J. McDonnell, Shawn Brisbay, Sherry Pierce, Hagop Kantarjian, Gary Rosner, Guillermo Garcia-Manero

From the Departments of Hematopathology, Biostatistics and Applied Mathematics, Molecular Pathology, and Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX

Address reprint requests to Guillermo Garcia-Manero, MD, Department of Leukemia, Box 428, The University of Texas M.D. Anderson Cancer Center, PO Box 301402, Houston, TX 77230-1402; e-mail: ggarciam{at}mdanderson.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: To study the relationship between protein expression and DNA methylation of a triad of cell-cycle regulatory genes known to be frequently methylated in adult acute lymphocytic leukemia (ALL).

PATIENTS AND METHODS: Protein expression of p73, p15, and p57^Kip2 was analyzed by immunohistochemistry using a tissue microarray (TMA) platform. The TMA was constructed using pretreatment bone marrow biopsy specimens from 64 adult patients with ALL. Protein expression was then correlated with DNA methylation and relevant clinical biologic characteristics.

RESULTS: p73 protein expression was observed in 19 (30%) patients, cytoplasmic p15 in 19 (31%), and p57 in 40 (70%). Three patients (5%) had expression of all three proteins, 16 (29%) of two proteins, 31 (55%) of one protein, and six (11%) of zero proteins. An inverse association was observed between p73 DNA methylation and protein expression (P = .003). This effect was not observed for either p15 or p57^Kip2. Expression of any of the proteins studied was not associated with any distinct biologic characteristic. By multivariate analysis, expression of p57^Kip2, cytoplasmic p15, or a combination of p57^Kip2 with either p15 or p73 was associated with a better overall survival (P < .001, .04, and .03 respectively).

CONCLUSION: Expression of a triad of cell cycle regulatory proteins that includes p73, p15, and p57^Kip2 has prognostic value in adult patients with ALL independently of the methylation status of each gene.

	INTRODUCTION

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DNA methylation of promoter-associated CpG islands is an epigenetic modification of DNA associated with gene silencing.¹ Aberrant DNA methylation of these promoter regions is a frequent event both in solid tumor and hematopoietic malignancies² and is considered a functional equivalent to the genetic alterations that characterize the process of malignant transformation. Although in vitro, this concept has been analyzed and validated in multiple models, there are no extensive studies of the relationship between aberrant DNA methylation and protein expression in patients with cancer. Such studies are needed because more genes are being reported as the target of aberrant DNA methylation, and diagnostic and prognostic assays with potential clinical use are being developed using these methylation markers.

To analyze the relationship between DNA methylation and protein expression in patients with leukemia, we have studied a cohort of patients with adult acute lymphocytic leukemia (ALL)³ characterized by the concordant methylation of multiple genes.³ In particular, aberrant DNA methylation of a triad of cell-cycle regulatory genes—p73, p15, and p57^Kip—has been shown to occur in close to 25% of these patients and to confer a poor prognosis to patients with Philadelphia chromosome (Ph) –negative disease.⁴ This group of patients thus provides a unique opportunity to explore the relationship between DNA methylation and protein expression of a group of frequently methylated genes with both biologic and clinical relevance.

To perform these studies, we developed a tissue microarray (TMA) platform constructed using pretreatment bone marrow biopsy specimens previously studied for DNA methylation of the above mentioned genes, and studied protein expression using immunohistochemical techniques. Subsequently, we correlated the presence of protein expression with the methylation status of each individual gene and studied the clinical and biologic significance of protein expression.

	PATIENTS AND METHODS

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Patient Characteristics
Sixty-four patients with ALL have been studied. Patient characteristics are summarized in Table 1. These patients corresponded to a group of 80 patients initially studied by us³ and were selected based on sample availability.

Tissue Microarray and Immunohistochemistry
A tissue arrayer device (Beecher Instruments, Silver Spring, MD) was used to construct the TMA. Hematoxylin and eosin–stained sections from each paraffin-embedded, formalin-fixed block were used to define diagnostic areas, and two random, representative 0.6-mm cores were obtained from each case and inserted in a grid pattern into a recipient paraffin block. Sections (5 µm) were then cut from each TMA and stained with antibodies to p73 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), p57 (1:75; NeoMarkers, Fremont, CA), and p15 (1:75; Santa Cruz Biotechnology). Following deparaffinization, heat-induced antigen retrieval techniques were used for each antibody. All sections were stained with a Dako Autostainer (Dako, Glostrup, Denmark) using standard immunohistochemical streptavidin-biotin techniques. Sections were first deparaffinized and then rehydrated. Antigens were retrieved using the Dako Target Retrieval solution in conjunction with steam. Sections were then treated to block endogenous peroxidase, avidin, biotin, and protein. All rinsing in between steps use a triethanolamine-buffered saline solution at pH 8.0. After blocking, sections were incubated with a primary antibody at the appropriate concentration for 60 minutes at room temperature (p15 and p57) or at 4°C overnight. Sections were rinsed and then incubated with a biotinylated universal secondary antibody followed by horseradish peroxidase–labeled streptavidin both at room temperature for 15 minutes each (Dako's LSAB+ kit). The 3,3 diaminobenzidine substrate was then applied for 15 minutes at room temperature. Sections were then counterstained with Mayer's hematoxylin, dehydrated, cleared, and coverslipped. The appropriate positive and negative controls were used for each antibody.

Each core was evaluated independently by two pathologists (C.B-R. and T.M.) for the percentage of tumor cell staining by blindly scoring a total of 500 cells in each of five randomly selected light microscopic fields. Disagreements were resolved by joint review on a multihead microscope. For each case, the core with the highest percentage of tumor cells stained was used for analysis. Images of the array were acquired using a Zeiss axiovert S100 inverted microscope (Zeiss, Thornwood, NY) and a Hamamatsu cooled CCD camera (Hamamatsu City, Japan). Images were then stored in an image archive linked to the tissue array database (TAD). The BLISS Image Analysis Workstation (Bacus Laboratories, Lombard, IL) was used to scan hematoxylin and eosin and immunohistochemically-stained TMA slides. Using the Webslide server, these slides were scored and entered directly into the TAD. Also, an Active X component in the TAD was developed that allows the image scanned on the BLISS system to be linked directly with the core in the TAD.

Analysis of DNA Methylation
DNA methylation of p73, p15, and p57^Kip2 in these samples has been previously reported.⁴ In summary, methylation of p15 and p57^Kip2 was analyzed using the combined bisulfite polymerase chain reaction (PCR) assay (COBRA),⁶ and p73 using methylation-specific PCR (MSP).⁷

Statistical Methods
The degree of protein expression was reflected in grades based on several parameters, including percentage of tumor present, degree of involvement, intensity, and localization. For the statistical analysis, a simple score (0 to 5) was obtaining by multiplying the percentage of tumor by the degree of involvement (1 to 3). A score of 1 or higher was considered as positive. This simplified system did not result in significant lost information. Statistical analysis was performed using the SAS software program (SAS version 8.1, Cary, NC) and S-Plus software program (S-Plus 2000, Insightful, Seattle, WA). We used Fisher's exact test to evaluate pair-wise relationships between categoric variables, such as gene expression and gene methylation. We used logistic regression to study the combined effects of characteristics on gene expression. We used the log-rank test to study the effects of individual characteristics on overall survival (OS) and disease-free survival (DFS). Graphs of survival probabilities are based on Kaplan-Meier estimates of survival distributions. The proportional hazards regression model allowed us to evaluate the combined effects of multiple factors on OS and DFS. We used stepwise model building with the proportional hazard model, requiring significance at the 0.25 level for a covariate to enter the model, and significance at the 0.10 level for it to remain. Effects were regarded as significant if the P value was <.05, and all P values are two-sided. All models were checked to satisfy the corresponding assumptions.

	RESULTS

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Patient Characteristics
Sixty-four patients were analyzed (Table 1). The median follow-up of the study group was 358 weeks, with the longest follow-up being 525 weeks. Out of these 64 patients, 59 (92%) responded to therapy, and 41 (69%) eventually relapsed. The median DFS was 67 weeks (95% CI, 43 to 114 weeks), and the median OS was 94 weeks (95% CI, 68 to 150 weeks). The characteristics of these patients differed from that of a group of adult patients with ALL (N = 202) treated with hyperCVAD during the same period of time at UTMDACC.⁵ In particular, 27% of the studied patients had Ph-positive disease compared to 16% of the comparative group (P = .04).

Frequency of p73, p15, and p57^Kip2 Protein Expression in Adult Patients With ALL
p73 immunostaining was observed in 19 (30%) of 62 assessable patients, and p57 in 40 (70%) of 57 patients. All patients had evidence of either nuclear or cytoplasmic p15 staining. Cytoplasmic p15 staining was observed in 19 (31%) of 61 patients (Fig 1). Of 56 patients for whom there were data for all three proteins, six patients (11%) had no evidence of protein expression of any of the three proteins, 31 (55%) had expression of only one protein, 16 (29%) of two proteins, and three patients (5%) of all three proteins.

View larger version (98K): [in this window] [in a new window]   Fig 1. Representative histologic and immunohistochemistry results. (A) High-power view of bone marrow tissue microarray showing sheets of blasts (hematoxylin & eosin stain); (B) p15 immunostain shows predominantly cytoplasmic staining in the majority of the blasts; (C) p57 immunostain demonstrates nuclear p57Kip2 immunopositivity in a subset of the blasts; (D) p73 immunostain highlights nuclear positivity in few blasts.

Association Between Protein Expression and DNA Methylation of p73, p15, and p57^Kip2
We had previously studied the methylation status of each of these genes in this cohort of patients.⁴ p57^Kip2 methylation ranged from 0% to 71% and was 15% in 17 (28%) of 59 patients. For p15, the range of methylation was 0% to 72% and was 15% in 17 (27%) of 64 patients. For p73 we used a nonquantitative method⁷ and methylation was observed in 15 (23%) of 64 patients. The association between methylation of each individual gene and protein expression is summarized in Table 2. In summary, a significant inverse association was observed between methylation of p73 and protein expression. Of the 15 patients with p73 DNA methylation, none expressed the protein, and none of the 19 patients with p73 protein expression had p73 methylation (P = .003). This effect was not observed for either p15 or p57. For p15 cytoplasmic expression, of 17 patients with p15 methylation, four patients expressed p15 protein and 13 did not (P = .5). Cytoplasmic p15 expression was studied instead of nuclear staining for this analysis because of its prognostic relevance. For p57^Kip2, of 15 patients with p57^Kip2 methylation, 10 expressed the protein and five did not (P = .2). No joint methylation effect was observed between methylation of any possible combination of the genes studied here and protein expression (data not shown). Figure 2 represents the methylation and protein expression patterns of these patients.

View larger version (13K): [in this window] [in a new window]   Fig 2. DNA methylation and protein expression profiles. (A) DNA methylation profiles. Each column represents a gene (p73M, p15M, p57Kip2M) and each row a patient. Black indicates methylation (ie, 15% for p15 and p57Kip2 or methylation-specific polymerase chain reaction positivity for p734). (B) Protein expression profile. Each column represents a protein (p73EX, p15EX, p57Kip2EX), and each row a patient. Black indicates lack of protein expression. Gray indicates it was not done.

Effects on overall survival By univariate analysis, and as expected, the presence of the Ph chromosome (P = .001), age as continuous variable (P = .004), and WBC count (P = .01)—also as a continuous variable—were associated with a worse OS. In contrast, the presence p57^Kip2 protein, regardless of localization (nuclear v cytoplasmic),was associated with a better outcome (P = .05, hazard ratio [HR], 0.526; 95% CI, 0.274 to 1.007). The median survival of patients with p57^Kip2 expression was 138 weeks compared with 68 weeks for those patients without it (Fig 3). No other variable analyzed including p73, p15, or a combination of proteins (the presence of p57^Kip2 with either p73 or cytoplasmic p15, herein protein-3) had prognostic value. Subsequently, we performed a multivariate analysis in 46 (71%) of the 64 patients for whom we had data for all 19 covariates detailed above. Because the sample size was too small to analyze all 19 covariates simultaneously, we performed a stepwise selection procedure. Protein-3 was not included in the initial stepwise selection procedure because it was derived from other variables. Seven covariates were retained for the Cox proportional hazard model (Table 3). These results indicate that patients with p57^Kip2 protein accumulation had about one eighth of the risk of death as that of a patient without p57^Kip2 protein expression (P < .001). Patients with cytoplasmic p15 expression had about one half of the risk of death as that of patients with p15 nuclear localization (P = .04). Because of the additive effect of methylation of multiple genes in ALL, we then analyzed the effect of the simultaneous expression of p73, p57, and p15 on survival. To do so, we replaced covariates p57^Kip2 and p15 from the model by p57 protein expression with p73 or cytoplasmic p15 expression (protein-3). These patients also had a significantly better outcome compared to patients without this pattern (HR, 0.393; 95% CI, 0.166 to 0.932; P = .03).

View larger version (16K): [in this window] [in a new window]   Fig 3. Impact on (A) overall survival and (B) disease-free survival of p57 protein expression.

In a multivariate analysis, three covariates were retained in the Cox proportional hazard model: the presence of the Ph abnormality, WBC, and hemoglobin. No effect was observed regarding p73, p15, or protein-3. Patients with p57^Kip2 expression had a slower relapse rate (HR, 0.692; 95% CI, 0.327 to 1.468; P = .33). No effect on DFS for any of the proteins studied here was observed when patients with Ph-positive disease were excluded from the analysis.

	DISCUSSION

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Aberrant DNA methylation is a very common phenomenon in leukemia, in particular in both pediatric and adult ALL.^3,8,9 As demonstrated by several independent studies, a significant subset of patients with ALL is characterized by the concordant methylation of multiple promoter CpG islands.^3,10 These methylation patterns do not seem to be the result of an stochastic process, as genes that are frequently methylated in other malignancies, such as p16,¹¹ are rarely methylated in these patients. Moreover, these methylation abnormalities are stable at the time of relapse in a majority of patients,¹² suggesting that aberrant DNA methylation is closely associated with the re-emergence of the leukemic clone.

We had previously identified that methylation of a cell-cycle regulatory pathway composed of p73, p15, and p57^Kip2 identified a subset of patients with Ph-negative disease and poor prognosis.⁴ Patients with methylation of less than two genes of this triad had an overall survival of 467 weeks versus 50 weeks for those with methylation of more than one gene (P = .02).⁴ Epigenetic inactivation of these three genes could have important implications in cell cycle control. p73 is a p53-like gene that has been found to be methylated in leukemias and lymphomas^7,13 and is functionally upstream of p57^Kip2.¹⁴ p15 and p57^Kip2 are two cyclin-dependent kinase inhibitors with a role in controlling cell cycle progression.^15,16 Both genes have been also shown to be the target of aberrant methylation in leukemia.^11,17-19 Of importance, knock-out animals of each individual gene have not shown a fully dominant malignant phenotype,^4,20,21 indicating that there is a high degree of redundancy in cell-cycle control systems.^22,23 Therefore, it is plausible that epigenetic inactivation of more than one gene on this pathway is required to induce a worse malignant phenotype. Aberrant methylation of these genes seems also to be a specific phenomenon, as other functionally related genes such as p53, p21, p16, or p14 have not been found to be the frequent target of aberrant methylation in these patients at initial presentation.^4,24 Of importance, clinically, methylation of this pathway is associated with differences in cell-cycle status: the median S+G2/M fraction was 9% (range, 2% to 19%) for patients with methylation of zero or one gene (n = 20 patients), compared with 14% (range, 4% to 30%; P = .04) for those with methylation of two or three genes (n = 8; Garcia-Manero, unpublished data).

Despite the importance of DNA methylation in cancer, very few studies have analyzed in vivo the relationship between DNA methylation and protein expression. This is of importance, as obviously DNA methylation is not the only mechanism that controls gene expression, and because most frequently used methylation assays cannot detect the presence of deletion/mutations.

To further explore the relationship between DNA methylation and protein expression, we developed a TMA platform using bone marrow specimens from a group of patients with ALL that had been extensively studied for DNA methylation by us, and that had been homogeneously treated.^3,5 This model offered a unique opportunity to study the relationship between DNA methylation and protein expression of a triad of genes frequently methylated and with clinical and biologic relevance. Indeed, to our knowledge, this is the first of such systematic studies, at least in leukemia.

Our results indicate that in vivo, the relation between DNA methylation and gene silencing/expression is gene specific. Of the three genes studied, only for p73 was there a significant inverse association between DNA methylation and protein expression. This phenomenon could be explained in several ways. First, the sensitivity/specificity of the assays used to study both protein expression and DNA methylation are not fully known and this could have an obvious effect in the interpretation of these results; second, the DNA methylation assays used do not detect deletions or inactivating mutations, known to occur at least for the p15 gene, that may also result in gene inactivation; third, DNA methylation may be monoallelic and may not be associated with gene silencing; and finally, with the DNA methylation assays used, it is not possible to assess whether DNA methylation is restricted to a subset of neoplastic cells, and its possible that this potential cellular heterogeneity may result in a discordance between DNA methylation and protein expression.

Despite the lack of a universal association between DNA methylation and protein expression, both the analysis of DNA methylation and protein expression indicate that lack of expression of a set of genes/proteins of a cell-cycle regulatory pathway allows the identification of a subset of patients with distinct prognosis. That being said, the analysis of protein expression is a more robust prognostic marker than that of DNA methylation, perhaps because it may be a more global assay than the detection of a methylation mark. Therefore, the results shown here have important clinical implications, as they confirm the prognostic value of the triad of genes/proteins studied, at least for patients with adult ALL treated with hyperCVAD-based chemotherapy. This information may allow the identification of a subset of patients with poor-risk Ph-negative disease that may benefit from early allogeneic stem-cell transplantation, as is currently done for patients with Ph-positive disease, or novel combination therapies including DNA hypomethylating agents.

There are several limitations to this study. The most important include the relative small sample size and the large number of covariates used. These results, therefore, should be considered as exploratory, and need to be confirmed in a larger subset of patients, and in different patient populations: adult patients treated with non–hyperCVAD-based chemotherapy and in high-risk pediatric patients.

In summary, our results indicate that the in vivo relationship between DNA methylation and protein expression is gene specific, and that caution is needed when extrapolating the functional role of DNA methylation in human cancer. Our results also indicate that the in vivo inactivation or proteins involved in cell cycle control, such as p73, p15, and p57^Kip2, have an important role in adult ALL.

	Authors' Disclosures of Potential Conflicts of Interest

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The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant/Advisory Role: Gary Rosner, Neopharm. For a detailed description of this category, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.

	Acknowledgment

 
We are grateful to Remigio Lopez for excellent technical support.

	NOTES

 
Funded in part by a Career Development Award from the American Society of Clinical Oncology, the Physician-Scientist Award from The University of Texas M.D. Anderson Cancer Center, and grants CA105771 and CA100067 from the National Institutes of Health (G.G-M.).

Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.

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

	REFERENCES

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Submitted October 28, 2004; accepted February 10, 2005.

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