Journal of Surgical Oncology 2013;108:374–377
Prognostic Impact of p300 Expression in Patients With Colorectal Cancer JUNG WOOK HUH, MD, PhD,1 HEE CHEOL KIM, MD, PhD,1* SEOK HYUNG KIM, MD, PhD,2 YOON AH PARK, MD,1 YONG BEOM CHO, MD, PhD,1 SEONG HYEON YUN, MD, PhD,1 WOO YONG LEE, MD, PhD,1 AND HO-KYUNG CHUN, MD, PhD3 1 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Department of Surgery, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea 2
Background: This study evaluated the expression of p300 in colorectal cancer, its relationship with clinicopathological characteristics, and its potential prognostic significance. Methods: The expression of p300 was measured using immunohistochemistry in tumors and surrounding normal mucosa from 199 patients with primary colorectal cancer. The patients were followed for a median period of 83 months. Results: Nuclear p300 expression was significantly associated with histology (P ¼ 0.031) and lymph node involvement (P ¼ 0.019). When the low and high p300 groups were subdivided according to tumor location, the disease‐free survival rate differed only for the patients with colon cancer (P ¼ 0.008). In addition, the disease‐free survival significantly differed with p300 expression for stage II disease (P ¼ 0.038), but not for stage III disease. Multivariate analysis revealed that lymph node involvement (P ¼ 0.014) and p300 expression (P ¼ 0.032) were independent predictors of overall survival in adenocarcinomas. Conclusion: The overexpression of p300 may be an independent favorable prognostic factor for disease‐free survival in patients with colorectal cancer. J. Surg. Oncol. 2013;108:374–377. ß 2013 Wiley Periodicals, Inc.
KEY WORDS: p300; colorectal cancer; immunohistochemistry
INTRODUCTION The prognosis of colorectal cancer is largely determined by the tumor‐node‐metastasis (TNM) stage and potential residual disease after initial surgery. Although the TNM classification is useful for staging patients and selecting specific treatments, it is limited because many patients at the same stage may have different clinical outcomes and different responses to adjuvant therapy. Therefore, novel tissue‐based prognostic indicators are necessary for developing new molecular‐level therapeutic approaches for colorectal cancer. The p300 protein is a transcriptional co‐activator with intrinsic histone acetyltransferase activity. It is involved in a wide array of cellular activities, such as DNA repair, cell growth, differentiation, apoptosis, and migration [1–3]. The clinical significance of p300 overexpression in colorectal cancer remains unclear [4–7]. A few studies have reported that the expression of p300 is associated with advanced stages of the disease and worse prognosis in colon cancer [4], while others found a significant role for p300 as a tumor suppressor in colon cancer [6,7]. In this study, we examined the expression of p300 in primary colorectal cancer using immunohistochemistry, its relationship with clinicopathological characteristics, and its potential prognostic significance.
PATIENTS AND METHODS One hundred ninety‐nine consecutive patients with primary colorectal adenocarcinoma and pathologic stage II and III disease who underwent surgical resection at our institution between March 2003 and February 2005 were selected for this study, on the basis of the availability of resected tissue. None of the patients had received neoadjuvant radiotherapy or chemotherapy prior to surgery. The non‐ necrotic portions of the tumors and the surrounding mucosa at a standard distance of 2 cm from the tumors were processed using the standard
ß 2013 Wiley Periodicals, Inc.
paraffin wax technique after fixation in 10% formalin for 24 hr. Representative sections were stained with hematoxylin and eosin (H&E). This study was approved by our Institutional Review Board.
Immunohistochemistry p300 expression was evaluated by immunohistochemical staining. Immunohistochemisty was performed on formalin‐fixed, paraffin‐ embedded tissue. A 3‐mm punch was taken from each paraffin‐ embedded block for tissue microarrays as described by Hendriks et al. [8]. Each section of normal and tumor tissues (4‐mm thick) was assessed by tissue microarray and was mounted on a glass slide. The tissue microarray slides were deparaffinized, and the tissues were then rehydrated with xylene and ethanol. The slides were washed with water and phosphate‐buffered saline (PBS). Endogenous peroxidase activity was blocked by incubating sections in 3% H2O2 in PBS for 30 min. The slides were then washed with water, after which heat‐induced epitope retrieval was performed. The slides derived from normal and tumor tissues (4‐mm thick) were stained with mouse monoclonal antibodies specific for p300 (clone DO‐7, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA). The annotation process included an estimation of the
Grant sponsor: Samsung Biomedical Research Institute; Grant number: SBRI‐CB11093. *Correspondence to: Hee Cheol Kim, MD, PhD, Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon‐ro, Gangnam‐gu, Seoul 135‐710, Korea. Fax: þ82‐2‐3410‐6980. E‐ mail: [email protected] Received 29 March 2013; Accepted 12 July 2013 DOI 10.1002/jso.23405 Published online 23 September 2013 in Wiley Online Library (wileyonlinelibrary.com).
p300 in Colorectal Cancer
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Fig. 1. Immunohistochemical expression of p300. A: Low expression and (B) high expression. intensity of immunoreactivity for a nuclear p300 (negative, 0; weak, 1; moderate, 2; strong, 3) and the fraction (%) of p300‐positive cells (75%, 4) (Fig. 1A and B). The total score was calculated as intensity multiplied by fraction. All tumors were categorized as high or low p300‐expressing based on a cut‐off score of 6. Normal colonic epithelium adjacent to tumor and lymphocytes served as internal negative controls. All histological slides were examined by two experienced gastrointestinal pathologists unaware of the clinical data or disease outcome. If there was a discrepancy in individual scores, both pathologists re‐evaluated the slides together to reach a consensus before combining the individual scores.
MSI Analysis The chromosomal DNA from the microdissected paraffin‐embedded tissue was crudely isolated using xylene/ethanol deparaffinization. MSI was determined by polymerase chain reaction (PCR) analysis using a DNA autosequencer (Applied Biosystems 373A sequencer; Applied Biosystems, Foster City, CA). Five Bethesda microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) were used to analyze the paired normal and tumor DNA for MSI, as described previously [9]. Tumors were classified as MSI‐high when at least two of the five loci showed MSI [9].
Statistical Analysis Statistical evaluation was carried out using the statistical package SPSS for Windows (Version 14.0; SPSS, Chicago, IL). p300 immunostaining was associated with the clinicopathological features using Student’s t‐test and the chi‐square test as appropriate. Disease‐free survival curves were calculated using the Kaplan–Meier method and differences between curves were evaluated using the log‐rank test. To identify the significant independent prognostic factors associated with disease‐free survival, the variables with P‐values less than 0.2 on univariate analysis were entered into a multivariate analysis performed using stepwise logistic regression. A value of P < 0.05 was considered statistically significant.
RESULTS The characteristics of the study population are shown in Table I. The subjects consisted of 111 men (55.8%), with a median age of 59 years (range 29–84) and a median tumor size of 5.0 cm (range 1–10). Using the 7th UICC TNM staging system, 108 and 91 patients had stage II and stage III cancers, respectively. The relationships between tumor p300 expression and the clinicopathological features of colorectal adenocarcinoma are summarized in Table II. The expression of p300 in tumors was Journal of Surgical Oncology
significantly associated with histology (P ¼ 0.031) and lymph node involvement (P ¼ 0.019). No significant association was detected between p300 expression and age, sex, tumor location, tumor size, depth of invasion, angiolymphatic invasion, preoperative level of serum carcinoembryonic antigen (CEA), adjuvant chemotherapy, or MSI status (Table II). During the follow‐up of a median of 83 months (range, 10–110 months), a significant difference was observed between disease‐free survival and the level of p300 expression (P ¼ 0.042). When the low and high p300 groups were subdivided according to tumor location, the disease‐free survival rate differed between the two groups only for the patients with colon cancer (P ¼ 0.008; Fig. 2A). For rectal cancers, the disease‐free survival rate did not differ between the two groups (P ¼ 0.800; Fig. 2B). The prognostic significance of p300 expression for survival according to the TNM stage is shown in Figure 3. The disease‐ free survival differed according to p300 expression for stage II and stage III disease, but a significant difference was only observed for patients with stage II cancer (P ¼ 0.038; Fig. 3A). On univariate analysis, the factors associated with poorer disease‐free survival were lymph node involvement (P ¼ 0.050) and p300 expression (P ¼ 0.042); however, age (P ¼ 0.394), sex (P ¼ 0.198), tumor location (P ¼ 0.892), histology (P ¼ 0.643), tumor size (P ¼ 0.814), depth of invasion (P ¼ 0.241), angiolymphatic invasion (P ¼ 0.107), preoperative level of serum CEA (P ¼ 0.379), adjuvant chemotherapy (P ¼ 0.779), or MSI status (P ¼ 0.665) were not significant. A multivariate analysis including univariate predictor variables with P values of less than 0.2 revealed that lymph node involvement (P ¼ 0.014) and p300 expression
TABLE I. Patients Characteristics (n ¼ 199) No. (%) Median age, years (range) Sex (M:F) Median tumor diameter, cm (range) Location Colon Rectum Tumor histology Well differentiated Moderately differentiated Poorly differentiated Mucinous TNM stage II III Angiolymphatic invasion Mean preoperative CEA, ng/ml (range) CEA, carcinoembryonic antigen.
59 (29–84) 111:88 5.0 (1.0–10.0) 143 (71.9) 56 (28.1) 21 (10.6) 158 (79.4) 13 (6.5) 7 (3.5) 108 (54.3) 91 (45.7) 46 (23.1) 4.3 9.4 (0.1–103.7)
376
Huh et al. (P ¼ 0.032) were independent predictors of disease‐free survival in patients with colorectal cancer (Table III).
TABLE II. Correlation Between a p300 Expression and the Clinicopathological Parameters p300 expression
DISCUSSION Parameter
N
Age, years
Prognostic Impact of p300 Expression in Patients With Colorectal Cancer JUNG WOOK HUH, MD, PhD,1 HEE CHEOL KIM, MD, PhD,1* SEOK HYUNG KIM, MD, PhD,2 YOON AH PARK, MD,1 YONG BEOM CHO, MD, PhD,1 SEONG HYEON YUN, MD, PhD,1 WOO YONG LEE, MD, PhD,1 AND HO-KYUNG CHUN, MD, PhD3 1 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea 3 Department of Surgery, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea 2
Background: This study evaluated the expression of p300 in colorectal cancer, its relationship with clinicopathological characteristics, and its potential prognostic significance. Methods: The expression of p300 was measured using immunohistochemistry in tumors and surrounding normal mucosa from 199 patients with primary colorectal cancer. The patients were followed for a median period of 83 months. Results: Nuclear p300 expression was significantly associated with histology (P ¼ 0.031) and lymph node involvement (P ¼ 0.019). When the low and high p300 groups were subdivided according to tumor location, the disease‐free survival rate differed only for the patients with colon cancer (P ¼ 0.008). In addition, the disease‐free survival significantly differed with p300 expression for stage II disease (P ¼ 0.038), but not for stage III disease. Multivariate analysis revealed that lymph node involvement (P ¼ 0.014) and p300 expression (P ¼ 0.032) were independent predictors of overall survival in adenocarcinomas. Conclusion: The overexpression of p300 may be an independent favorable prognostic factor for disease‐free survival in patients with colorectal cancer. J. Surg. Oncol. 2013;108:374–377. ß 2013 Wiley Periodicals, Inc.
KEY WORDS: p300; colorectal cancer; immunohistochemistry
INTRODUCTION The prognosis of colorectal cancer is largely determined by the tumor‐node‐metastasis (TNM) stage and potential residual disease after initial surgery. Although the TNM classification is useful for staging patients and selecting specific treatments, it is limited because many patients at the same stage may have different clinical outcomes and different responses to adjuvant therapy. Therefore, novel tissue‐based prognostic indicators are necessary for developing new molecular‐level therapeutic approaches for colorectal cancer. The p300 protein is a transcriptional co‐activator with intrinsic histone acetyltransferase activity. It is involved in a wide array of cellular activities, such as DNA repair, cell growth, differentiation, apoptosis, and migration [1–3]. The clinical significance of p300 overexpression in colorectal cancer remains unclear [4–7]. A few studies have reported that the expression of p300 is associated with advanced stages of the disease and worse prognosis in colon cancer [4], while others found a significant role for p300 as a tumor suppressor in colon cancer [6,7]. In this study, we examined the expression of p300 in primary colorectal cancer using immunohistochemistry, its relationship with clinicopathological characteristics, and its potential prognostic significance.
PATIENTS AND METHODS One hundred ninety‐nine consecutive patients with primary colorectal adenocarcinoma and pathologic stage II and III disease who underwent surgical resection at our institution between March 2003 and February 2005 were selected for this study, on the basis of the availability of resected tissue. None of the patients had received neoadjuvant radiotherapy or chemotherapy prior to surgery. The non‐ necrotic portions of the tumors and the surrounding mucosa at a standard distance of 2 cm from the tumors were processed using the standard
ß 2013 Wiley Periodicals, Inc.
paraffin wax technique after fixation in 10% formalin for 24 hr. Representative sections were stained with hematoxylin and eosin (H&E). This study was approved by our Institutional Review Board.
Immunohistochemistry p300 expression was evaluated by immunohistochemical staining. Immunohistochemisty was performed on formalin‐fixed, paraffin‐ embedded tissue. A 3‐mm punch was taken from each paraffin‐ embedded block for tissue microarrays as described by Hendriks et al. [8]. Each section of normal and tumor tissues (4‐mm thick) was assessed by tissue microarray and was mounted on a glass slide. The tissue microarray slides were deparaffinized, and the tissues were then rehydrated with xylene and ethanol. The slides were washed with water and phosphate‐buffered saline (PBS). Endogenous peroxidase activity was blocked by incubating sections in 3% H2O2 in PBS for 30 min. The slides were then washed with water, after which heat‐induced epitope retrieval was performed. The slides derived from normal and tumor tissues (4‐mm thick) were stained with mouse monoclonal antibodies specific for p300 (clone DO‐7, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA). The annotation process included an estimation of the
Grant sponsor: Samsung Biomedical Research Institute; Grant number: SBRI‐CB11093. *Correspondence to: Hee Cheol Kim, MD, PhD, Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon‐ro, Gangnam‐gu, Seoul 135‐710, Korea. Fax: þ82‐2‐3410‐6980. E‐ mail: [email protected] Received 29 March 2013; Accepted 12 July 2013 DOI 10.1002/jso.23405 Published online 23 September 2013 in Wiley Online Library (wileyonlinelibrary.com).
p300 in Colorectal Cancer
375
Fig. 1. Immunohistochemical expression of p300. A: Low expression and (B) high expression. intensity of immunoreactivity for a nuclear p300 (negative, 0; weak, 1; moderate, 2; strong, 3) and the fraction (%) of p300‐positive cells (75%, 4) (Fig. 1A and B). The total score was calculated as intensity multiplied by fraction. All tumors were categorized as high or low p300‐expressing based on a cut‐off score of 6. Normal colonic epithelium adjacent to tumor and lymphocytes served as internal negative controls. All histological slides were examined by two experienced gastrointestinal pathologists unaware of the clinical data or disease outcome. If there was a discrepancy in individual scores, both pathologists re‐evaluated the slides together to reach a consensus before combining the individual scores.
MSI Analysis The chromosomal DNA from the microdissected paraffin‐embedded tissue was crudely isolated using xylene/ethanol deparaffinization. MSI was determined by polymerase chain reaction (PCR) analysis using a DNA autosequencer (Applied Biosystems 373A sequencer; Applied Biosystems, Foster City, CA). Five Bethesda microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) were used to analyze the paired normal and tumor DNA for MSI, as described previously [9]. Tumors were classified as MSI‐high when at least two of the five loci showed MSI [9].
Statistical Analysis Statistical evaluation was carried out using the statistical package SPSS for Windows (Version 14.0; SPSS, Chicago, IL). p300 immunostaining was associated with the clinicopathological features using Student’s t‐test and the chi‐square test as appropriate. Disease‐free survival curves were calculated using the Kaplan–Meier method and differences between curves were evaluated using the log‐rank test. To identify the significant independent prognostic factors associated with disease‐free survival, the variables with P‐values less than 0.2 on univariate analysis were entered into a multivariate analysis performed using stepwise logistic regression. A value of P < 0.05 was considered statistically significant.
RESULTS The characteristics of the study population are shown in Table I. The subjects consisted of 111 men (55.8%), with a median age of 59 years (range 29–84) and a median tumor size of 5.0 cm (range 1–10). Using the 7th UICC TNM staging system, 108 and 91 patients had stage II and stage III cancers, respectively. The relationships between tumor p300 expression and the clinicopathological features of colorectal adenocarcinoma are summarized in Table II. The expression of p300 in tumors was Journal of Surgical Oncology
significantly associated with histology (P ¼ 0.031) and lymph node involvement (P ¼ 0.019). No significant association was detected between p300 expression and age, sex, tumor location, tumor size, depth of invasion, angiolymphatic invasion, preoperative level of serum carcinoembryonic antigen (CEA), adjuvant chemotherapy, or MSI status (Table II). During the follow‐up of a median of 83 months (range, 10–110 months), a significant difference was observed between disease‐free survival and the level of p300 expression (P ¼ 0.042). When the low and high p300 groups were subdivided according to tumor location, the disease‐free survival rate differed between the two groups only for the patients with colon cancer (P ¼ 0.008; Fig. 2A). For rectal cancers, the disease‐free survival rate did not differ between the two groups (P ¼ 0.800; Fig. 2B). The prognostic significance of p300 expression for survival according to the TNM stage is shown in Figure 3. The disease‐ free survival differed according to p300 expression for stage II and stage III disease, but a significant difference was only observed for patients with stage II cancer (P ¼ 0.038; Fig. 3A). On univariate analysis, the factors associated with poorer disease‐free survival were lymph node involvement (P ¼ 0.050) and p300 expression (P ¼ 0.042); however, age (P ¼ 0.394), sex (P ¼ 0.198), tumor location (P ¼ 0.892), histology (P ¼ 0.643), tumor size (P ¼ 0.814), depth of invasion (P ¼ 0.241), angiolymphatic invasion (P ¼ 0.107), preoperative level of serum CEA (P ¼ 0.379), adjuvant chemotherapy (P ¼ 0.779), or MSI status (P ¼ 0.665) were not significant. A multivariate analysis including univariate predictor variables with P values of less than 0.2 revealed that lymph node involvement (P ¼ 0.014) and p300 expression
TABLE I. Patients Characteristics (n ¼ 199) No. (%) Median age, years (range) Sex (M:F) Median tumor diameter, cm (range) Location Colon Rectum Tumor histology Well differentiated Moderately differentiated Poorly differentiated Mucinous TNM stage II III Angiolymphatic invasion Mean preoperative CEA, ng/ml (range) CEA, carcinoembryonic antigen.
59 (29–84) 111:88 5.0 (1.0–10.0) 143 (71.9) 56 (28.1) 21 (10.6) 158 (79.4) 13 (6.5) 7 (3.5) 108 (54.3) 91 (45.7) 46 (23.1) 4.3 9.4 (0.1–103.7)
376
Huh et al. (P ¼ 0.032) were independent predictors of disease‐free survival in patients with colorectal cancer (Table III).
TABLE II. Correlation Between a p300 Expression and the Clinicopathological Parameters p300 expression
DISCUSSION Parameter
N
Age, years
