Tumor Biol. (2014) 35:5735–5740 DOI 10.1007/s13277-014-1760-0

RESEARCH ARTICLE

Clinical significance of Ki-67 and p53 expression in curatively resected non-small cell lung cancer Hee Kyung Ahn & Minkyu Jung & Seung-Yeon Ha & Jae-Ik Lee & Inkeun Park & Young Saing Kim & Junshik Hong & Sun Jin Sym & Jinny Park & Dong Bok Shin & Jae Hoon Lee & Eun Kyung Cho

Received: 29 November 2013 / Accepted: 13 February 2014 / Published online: 16 April 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract The aim of this study is to explore the association of Ki-67 and p53 expression with prognosis in non-small cell lung cancer (NSCLC) patients who underwent curative resection. We retrospectively identified 116 consecutive patients with stages I– III NSCLC who underwent curative resection at a single center from January 2007 to December 2012. Ki-67 and p53 expression was assessed by immunohistochemistry. Data on clinicopathologic features and survival were collected retrospectively. Ki-67 expression in 109 samples and p53 expression in 115 patients were analyzed. According to the results, 108 patients (99 %) showed at least some expression of Ki-67. The median Ki-67 expression level was 30 %. Positive p53 expression was observed in 91 (79 %) patients. Higher Ki-67 expression (>40 %) was significantly more frequent in male (26 vs. 4 % in female, p=0.002), ever-smoker (31 vs. 10 % in never-smoker, p=0.024), and non-adenocarcinoma (30 vs. 11 % of adenocarcinoma, p=0.012) patients. In univariable analysis, median disease-free survival (DFS) was shorter with higher Ki-67 expression (16.1 vs. 61.9 months in those with lower Ki-67 Hee Kyung Ahn and Minkyu Jung contributed equally to this journal. H. K. Ahn : M. Jung : I. Park : Y. S. Kim : J. Hong : S. J. Sym : J. Park : D. B. Shin : J. H. Lee : E. K. Cho (*) Division of Hematology and Oncology, Department of Internal Medicine, Gachon University Gil Medical Center, 1198 Guwol-dong, Namdong-gu, Incheon 405-760, Republic of Korea e-mail: [email protected] M. Jung Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea S.40 %) showed an independent association with shorter DFS in NSCLC patients who underwent curative resection. Keywords Non-small cell lung carcinoma . Ki-67 . p53 . Immunostaining . Resection . Prognosis

Introduction More than half of patients with non-small cell lung cancer (NSCLC) suffer from recurrence after curative resection and adjuvant treatment. Therefore, additional prognostic factors other than TNM stage are needed in order to refine the prognosis in NSCLC patients who received treatment with curative intent. Ki-67, a nuclear protein, is expressed during the active phases of the cell cycle, except G0 stage, and is therefore considered a marker of cell proliferation [1]. High growth fraction is a hallmark of malignancy. The association of high Ki-67 expression and poor prognosis has been investigated in various cancers. Ki-67 expression in lung tumor, including both primary lung cancer and lung metastases, showed an association with decreased local tumor progression-free survival and disease-specific survival after radiofrequency ablation [2]. Studies investigating the prognostic value of Ki-67 in NSCLC patients are heterogeneous in patient population, methodology, and cutoff value of Ki-67, resulting in ambiguous findings regarding the prognostic impact of Ki-67 in NSCLC [3].

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Table 1 Patients’ characteristics and association between Ki-67 and p53 expression Characteristics

Number (%) Total=116

Age (years) Median, range ≤60 >60 Sex Male Female Smoking history Smoker Never smoker Unknown Histology Adenocarcinoma Non-adenocarcinoma TNM Stage IA IB II III T stage T1 T2 T3–4 N stage N0 N1 N2 Adjuvant treatment Chemotherapy Radiotherapy Chemoradiotherapy Sequential RT-chemotherapy None

Ki-67 expression Number (%) Total=109

p53 expression Number (%) Total=115

Low ≤40 % 89 (82 %)

High >40 % 20 (18 %)

p value

65, 24–81 40 (35) 76 (65)

33 (89) 56 (78)

4 (11) 16 (22)

70 (60) 46 (40)

47 (72) 42 (96)

40 (35) 46 (40) 30 (26)

p value

Positive

Negative

91 (79 %)

24 (79 %)

0.194

32 (82) 59 (78)

7 (18) 17 (22)

0.636

18 (28) 2 (4)

0.002

56 (80) 35 (78)

14 (20) 10 (22)

0.817

27 (70) 38 (91)

12 (31) 4 (10)

0.024

35 (88) 35 (78)

5 (13) 10 (22)

0.270

70 (60) 46 (40)

59 (89) 30 (70)

7 (11) 13 (30)

0.012

57 (83) 34 (74)

12 (17) 12 (26)

0.349

24 (21)

21 (88)

3 (13)

0.840

16 (67)

8 (33)

0.323

34 (29) 32 (28) 26 (23)

24 (78) 24 (80) 20 (83)

7 (23) 6 (20) 4 (17)

27 (79) 26 (81) 22 (88)

7 (21) 6 (19) 3 (12)

37 (32) 68 (59) 11 (9)

31 (86) 49 (78) 9 (90)

5 (14) 14 (22) 1 (10)

0.562

26 (72) 54 (79) 11 (100)

10 (28) 14 (21) 0 (0)

0.152

65 (56) 32 (28) 19 (16)

48 (80) 27 (87) 14 (78)

12 (20) 4 (13) 4 (22)

0.665

49 (75) 27 (84) 15 (83)

16 (25) 5 (16) 3 (17)

0.596

60 (52) 2 (2) 1 (1) 1 (1) 52 (45)

46 (84) 1 (50) 1 (100) 1 (100) 40 (80)

9 (16) 1 (50) 0 (0) 0 (0) 10 (20)

0.534

46 (77) 2 (100) 0 (0) 1 (100) 42 (82)

14 (23) 0 (0) 1 (100) 0 (0) 9 (18)

0.449

p53 is a tumor suppressor nuclear protein, and p53 mutation is found in up to 50 % of patients with NSCLC [4]. Mutant p53 proteins have a longer half-life than wild-type p53 and therefore accumulate in the nucleus and can be detected by immunohistochemistry [5]. Overexpression of p53 by immunohistochemistry (IHC) or mutant p53 is associated with worse survival in patients with NSCLC [6–10], although current research on this topic is limited. Use of only one prognostic biomarker is limited, while consideration of known biomarkers together is more

powerful. This study investigated the prognostic impact of Ki-67 and p53 detected by IHC on disease-free survival (DFS) in stages I–III NSCLC patients who underwent curative resection.

Methods We retrospectively identified consecutive NSCLC patients who underwent curative surgical resection at Gachon University Gil

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Fig. 1 Ki-67 and p53 expression by immunochemistry (magnification ×400). a Negative Ki-67 expression, b high Ki-67 expression, c negative p53 expression, and d positive p53 expression

Medical Center, Incheon, Korea, from 2007 to 2012. Patients were included based on the following criteria: (1) histologic diagnosis of NSCLC; (2) complete removal of gross tumor; and

a

(3) TNM pathologic stage I, II, or III (American Joint Committee on Cancer seventh edition). Patients who received neoadjuvant chemotherapy or radiotherapy were excluded. Patients’ clinicopathologic data, including sex, age, smoking history, date of diagnosis, date of surgery, pathological stage, histology, adjuvant chemotherapy, date of death, and date of last followup, were collected from medical records. Chest contrast computed tomography scan was performed for assessment of progression or recurrence of lung cancer. This study was approved by the Institutional Review Board at Gachon University Gil Medical Center, and informed consent was waived. Ki-67 and p53 expression was measured by immunohistochemical staining. Monoclonal antibodies to p53 (1:50, DAKO, Glostrup, Denmark) and Ki-67 (1:50, DAKO, Glostrup, Denmark) were used. p53 was considered positive when more than 10 % of the tumor cell nuclei showed strong

b

Fig. 2 Kaplan-Meier survival plots by Ki-67 expression. a Disease-free survival and b overall survival

Fig. 3 Disease-free survival according to Ki-67 expression among 42 stage I patients who did not receive adjuvant chemotherapy

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staining with a dark brown color. For Ki-67 expression, the percentage of tumor cells stained in nuclei was assessed. The association between clinicopathologic characteristics and immunohistochemistry results was analyzed using the chi-square test or Fisher’s exact test, as appropriate. Maximally selected rank statistics using the Maxstat package in the open source statistical software R (R Development Core Team (2005) was used for determination of the cutoff point of Ki67 value [11]) were used. Disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan-Meier method and compared using the Breslow test. Cox proportional hazard regression modeling was used for assessment of hazard ratio and 95 % confidence interval of each variable on DFS. DFS was defined from the date of surgery to the date of radiologically documented recurrence of NSCLC, the last exam, or death from any cause. Overall survival was estimated as the time from the date of the surgical resection to the date of death or the last follow-up visit. Null hypotheses of no difference were rejected if p values were less than 0.05 or, equivalently, if the 95 % confidence intervals (CIs) of risk point estimates excluded one.

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clinicopathologic characteristics, including sex, age, TNM pathologic stage, and histology. Median DFS of the 116 patients was 61.9 months, and median OS was not reached. In univariable analysis, DFS or OS did not differ significantly according to p53 expression status. Higher Ki-67 expression (>40 %) showed an association with shorter DFS (median DFS 16 vs. 62 months in patients with Ki-67≤40 %, p=0.005) and poorer OS (2-year OS rate 75 vs. 90 % in patients with Ki-67≤40 %, p=0.027) (Fig. 2). Among 42 patients with pathologic stage I disease who did not receive adjuvant chemotherapy, patients with higher Ki-67 expression (>40 %, n=7) had worse DFS (2-year DFS rate 57 %) than patients with lower Ki-67 expression (2-year DFS rate 88 %) (p=0.02, Fig. 3). In multivariable analysis, we included sex, age, smoking history, Ki-67 expression, tumor histology, and TNM stage in order to test the association with DFS. Age≥60 years (HR 2.4, 95 % CI 1.1–5.2), higher TNM stage, and higher expression of Ki-67(>40 %) were independent prognostic factors for shorter DFS (Table 2).

Discussion Results A total of 116 patients with pathologic stage I, II, or III who underwent curative resection from 2007 to 2012 were identified. Patient characteristics are shown in Table 1. Four patients had a microscopically positive resection margin, and three of them received adjuvant radiotherapy. Adjuvant chemotherapy or radiotherapy was administered in 64 patients (56 %). Regarding the time of analysis, recurrence was diagnosed in 35 patients (30.2 %), and death occurred in 19 cases (16.4 %). The median observation period of living patients was 30 months (range 1–71 months). Ki-67 expression level was determined in 109 patients, and 108 patients showed at least some expression of Ki-67. In 20 patients, Ki-67 expression was less than 10 %. Median value of Ki-67 was 30 % (range 0–95 %). p53 expression was evaluated in 115 patients; 91 patients (79 %) showed positive p53 staining. Ki-67 expression level did not differ significantly according to p53 expression status (mean value of Ki-67 expression was 22 % in the negative p53 group vs. 30 % in the positive p53 group). Representative images of Ki-67 and p53 immunostaining results are shown in Fig. 1. The association of clinicopathologic features of the patients and immunostaining markers was examined. Higher Ki-67 expression (>40 %) was more common in males (26 vs. 4 % in females, p40 % Non-adenocarcinoma TNM Stage I II III

2.418 (1.120–5.217) 0.732 (0.314–1.708) 2.875 (1.326–6.234) 0.460 (0.193–1.100)

0.024 0.471 0.008 0.081

1 2.498 (1.114–5.598) 6.172 (2.675–14.238)

0.026