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Combination immunohistochemistry for SMAD4 and Runt-related transcription factor 3 may identify a favorable prognostic subgroup of pancreatic ductal adenocarcinomas

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Oncotarget. 2017; 8:76699-76711. https://doi.org/10.18632/oncotarget.20815

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Yangkyu Lee, Hyejung Lee, Hyunjin Park, Jin-Won Kim, Jin-Hyeok Hwang, Jaihwan Kim, Yoo-Seok Yoon, Ho-Seong Han and Haeryoung Kim _

Abstract

Yangkyu Lee1, Hyejung Lee2, Hyunjin Park2, Jin-Won Kim3, Jin-Hyeok Hwang3, Jaihwan Kim3, Yoo-Seok Yoon4, Ho-Seong Han4 and Haeryoung Kim2

1Department of Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea

2Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea

3Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea

4Department of Surgery, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea

Correspondence to:

Haeryoung Kim, email: [email protected]

Keywords: pancreatic ductal adenocarcinoma, SMAD4, RUNX3, immunohistochemistry, prognosis

Received: June 05, 2017     Accepted: August 23, 2017     Published: September 11, 2017

ABSTRACT

Purposes: SMAD4/DPC4 mutations have been associated with aggressive behavior in pancreatic ductal adenocarcinomas (PDAC), and it has recently been suggested that RUNX3 expression combined with SMAD4 status may predict the metastatic potential of PDACs. We evaluated the prognostic utility of SMAD4/RUNX3 status in human PDACs by immunohistochemistry.

Materials and Methods: Immunohistochemical stains were performed for SMAD4 and RUNX3 on 210 surgically resected PDACs, and the results were correlated with the clinicopathological features.

Results: Loss of SMAD4 expression was associated with poor overall survival (OS) (p = 0.015) and progression-free survival (PFS) (p = 0.044). Nuclear RUNX3 expression was associated with decreased OS (p = 0.010) and PFS (p = 0.009), and more frequent in poorly differentiated PDACs (p = 0.037). On combining RUNX3/SMAD4 status, RUNX3-/SMAD4+ PDACs demonstrated longer OS (p = 0.008, median time; RUNX3-/SMAD4+ 34 months, others 17 months) and PFS (p = 0.009, median time; RUNX3-/SMAD4+ 29 months, others 8 months) compared to RUNX3+/SMAD4+ and SMAD4- groups; RUNX3-/SMAD4+ was a significant independent predictive factor for both OS [p = 0.025, HR 1.842 (95% CI 1.079-3.143)] and PFS [p = 0.020, HR 1.850 (95% CI 1.100-3.113)].

Conclusions: SMAD4-positivity with RUNX3-negativity was a significant independent predictive factor for favorable OS and PFS in PDAC. This is the first and large clinicopathological study of RUNX3/SMAD4 expression status in human PDAC. Combination immunohistochemistry for SMAD4 and RUNX3 may help identify a favorable prognostic subgroup of PDAC.


INTRODUCTION

Pancreas cancer is the fourth most common cause of cancer-related deaths in industrialized countries, with an overall 5-year survival rate of about 8% [1]. The majority of patients are diagnosed at an advanced stage with distant metastasis or invasion of adjacent organs, and less than 20% of patients are amenable to curative resection [1].

The common genetic alterations in pancreatic carcinogenesis include activating mutations in KRAS, telomere shortening, and inactivation of CDK2NA/p16, TP53 and SMAD4 genes [2, 3]. However, the molecular landscape of pancreatic ductal adenocarcinomas (PDACs) is extremely vast. For example, recent large-scale genomic analyses on PDACs have identified various subtypes of these cancers highlighting their heterogeneity, e.g. the “Collisson class” (classical, quasimesenchymal and exocrine-like subtypes), “Moffitt tumor class” (basal-like versus classical), “Moffitt stromal class” (activated stroma, normal stroma) and the subtypes recently defined by Bailey et al. (squamous, pancreatic progenitor, immunogenic and aberrantly differentiated endocrine exocrine (ADEX) subtypes) [46]. Thus, targeting the specific molecular subtypes would ideally help improve the outcomes of PDAC patients, in the way that selected patients with breast and lung cancers have greatly benefitted from targeted therapy over the past decades.

While such high-throughput genomic analyses have yielded promising results, it is also important that inexpensive and easily applicable tests (e.g. immunohistochemical assays) are developed for predicting therapeutic response and prognosis. In this regard, Runt-related transcription factor 3 (RUNX3) is an interesting candidate biomarker for PDAC, as it has recently been demonstrated to play a role as a “metastatic switch” in PDACs in genetically engineered mouse models: RUNX3 was suggested to be an important determinant of the ability of PDAC cells to metastasize to distant sites or to proliferate locally [7, 8]. Moreover, RUNX3 expression was shown to be influenced by SMAD4 status in the same study: heterozygous deletion of SMAD4 (+/−) attenuated RUNX3 levels compared to intact SMAD4 (+/+) and homozygously-deleted SMAD4 (−/−) [8].

If the combination of RUNX3 and SMAD4 status could predict the behavior of PDAC, analyzing RUNX3 and SMAD4 protein expression status by immunohistochemistry in resected or biopsied PDAC tissues would greatly benefit pancreatic cancer patients by helping to guide treatment plans. For example, PDACs with increased metastatic potential could benefit from systemic chemotherapy, while the more locally proliferative PDACs would be candidates for more aggressive local treatment, such as surgery or radiotherapy. Therefore, in this study, we performed an immunohistochemical analysis of RUNX3 and SMAD4 expression in our cohort of 210 surgically resected PDACs, and compared the clinicopathological features of PDACs according to RUNX3/SMAD4 expression status.

RESULTS

Clinicopathological characteristics

The clinicopathological characteristics of 210 cases are summarized in Table 1. 126 (60%) out of 210 patients were male, and the median age at initial operation was 65 years (range: 29~89 years). Most patients underwent operation for initial treatment. Only 7 patients received neoadjuvant chemotherapy before resection. The majority of PDACs were moderately differentiated (159/210, 75.7%) and were pT3 by the American Joint Committee on Cancer (AJCC) 7th edition (201/210, 95.7%). When the recently published size-based AJCC 8th edition was applied, 12.4% (26/210) were pT1 and 58.1% (122/210) were pT2, while 28.5% and 1.0% were pT3 and pT4, respectively, at the time of surgery. 130/210 (61.9%) cases had lymph node metastasis. On follow up, 86/210 (41.0%) and 121/210 (57.6%) developed local recurrence and distant metastasis, respectively, and 174/210 (82.9%) patients were deceased at last follow up.

Table 1: Patient and tumor characteristics (n = 210)

Sex

 Male

126 (60.0%)

 Female

84 (40.0%)

Age at operation, median (range, years)

65 (29–89)

Initial CEA, median (ng/ml)

3.0 (0.01-50.6)

Initial CA19–9, median (IU/ml)

121.2 (0.01–10000)

Tumor size

 ≤ 2 cm

26 (12.4%)

 > 2 cm and ≤ 4 cm

123 (58.6%)

 > 4 cm

61 (29.0%)

Preoperative treatment

 None

203 (96.7%)

 Neoadjuvant chemotherapy

7 (3.3%)

Histologic grade

 Well differentiated

23 (11.0%)

 Moderately differentiated

159 (75.7%)

 Poorly differentiated

23 (11.0%)

 Undifferentiated

5 (2.3%)

pT stage (AJCC 7th Ed.)

 pT1

2 (1.0%)

 pT2

5 (2.3%)

 pT3

201 (95.7%)

 pT4

2 (1.0%)

pT stage (AJCC 8th Ed.)

 pT1 (≤ 2 cm)

26 (12.4%)

 pT1a (≤ 0.5 cm)

0

 pT1b (> 0.5 cm and <1 cm)

0

 pT1c (≥ 1 cm and ≤ 2 cm)

26

 pT2 (>2 cm and ≤ 4 cm)

122 (58.1%)

 pT3 (>4 cm)

60 (28.5%)

 pT4

2 (1.0%)

pN stage (AJCC 7th Ed.)

 pN0

80 (38.1%)

 pN1

130 (61.9%)

pN stage (AJCC 8th Ed.)

 pN0 (no metastasis)

80 (38.1%)

 pN1 (1~3)

97 (46.2%)

 pN2 (≥ 4)

33 (15.7%)

Local recurrence

 Absent

124 (59.0%)

 Present

86 (41.0%)

Distant metastasis

 Absent

89 (42.4%)

 Present

121 (57.6%)

Status at last follow up

 Alive

36 (17.1%)

 Deceased

174 (82.9%)

RUNX3 and SMAD4 expression in pancreatic ductal adenocarcinomas

The immunohistochemical stain results are demonstrated in Figure 1. Loss of SMAD4 expression (“SMAD4-”) was observed in 145 (72.5%) cases, while the remaining 55 (27.5%) cases had intact SMAD4 (“SMAD4+”). RUNX3 expression (“RUNX3+”) was identified in 121 cases (59.0%) compared with remaining 84 cases showing no RUNX3 expression (“RUNX3−”). On combining the SMAD4 and RUNX3 status, 25 (11.9%) PDACs demonstrated intact SMAD4 with no RUNX3 expression (RUNX3−/SMAD4+).

Immunohistochemical stain results for RUNX3 and SMAD4 in PDACs and normal pancreatic parenchyma.

Figure 1: Immunohistochemical stain results for RUNX3 and SMAD4 in PDACs and normal pancreatic parenchyma. (A) RUNX3 is not expressed in normal pancreatic tissue. A few scattered lymphocytes show nuclear RUNX3 expression. Representative PDACs with (B) no nuclear RUNX3 expression and (C) RUNX3 expression. (D) Intact SMAD4 expression in normal pancreatic tissue. PDACs with (E) SMAD4 loss, and (F) intact SMAD4 expression (original magnification x400).

When the clinicopathological features were compared according to SMAD4 and RUNX3 expression status (Table 2), we found SMAD4 loss was associated with increased tumor size (p = 0.030), higher pN stage (AJCC 8th ed.) (p = 0.035), poor histological differentiation (p = 0.051) and higher pT stage (AJCC 8th ed.) (p = 0.054), although the latter two parameters were marginally significant. When the disease recurrence patterns were compared, PDACs with SMAD4 loss tended to result more frequently in distant metastasis compared to SMAD4-intact PDACs (p = 0.058), while there was no difference in the frequency of local recurrence according to SMAD4 expression status. RUNX3 expression was associated with tumor size (p = 0.039) and poor histological differentiation (p = 0.033).

Table 2: RUNX3 and SMAD4 status and clinicopathological features in pancreatic ductal adenocarcinoma cases

Parameters

SMAD4 loss (SMAD4-)

Intact SMAD4 (SMAD4+)

p-value

No RUNX3 expression (RUNX3-)

RUNX3 expression (RUNX3+)

p-value

Sex

0.747

0.774

 Male

88 (60.7%)

32 (58.2%)

49 (58.3%)

73 (60.3%)

 Female

57 (39.3%)

23 (41.8%)

35 (41.7%)

48 (39.7%)

Age at operation (mean ± SD)

65.1 ± 9.94

62.3 ± 9.92

0.082

65.3 ± 8.61

63.3 ± 10.96

0.146

Tumor size

0.030

0.039

 ≤ 2 cm

12 (8.3%)

12 (21.8%)

8 (9.5%)

17 (14.0%)

 > 2 cm and ≤ 4 cm

90 (62.0%)

28 (50.9%)

58 (69.0%)

62 (51.2%)

 > 4 cm

43 (29.7%)

15 (27.3%)

18 (21.4%)

42 (34.7%)

Histologic grade

0.051

0.033

 WD/MD

122 (84.1%)

52 (94.5%)

78 (92.9%)

100 (82.6%)

 PD/UD

23 (15.9%)

3 (5.5%)

6 (7.1%)

21 (17.4%)

Lymphatic invasion

0.665

0.973

 Absent

81 (56.3%)

29 (52.7%)

46 (54.8%)

66 (55.0%)

 Present

63 (43.7%)

26 (47.3%)

38 (45.2%)

54 (45.0%)

Venous invasion

0.591

0.228

 Absent

91 (63.2%)

37 (67.3%)

58 (69.0%)

73 (60.8%)

 Present

53 (36.8%)

18 (32.7%)

26 (31.0%)

47 (39.2%)

Perineural invasion

0.850

0.926

 Absent

22 (15.3%)

9 (16.4%)

13 (15.5%)

18 (15.0%)

 Present

122 (84.7%)

46 (83.6%)

71 (84.5%)

102 (85.0%)

pT stage (AJCC 7th Ed.)

0.725

0.509

 pT1

1 (0.7%)

0 (0.0%)

1 (1.2%)

0 (0.0%)

 pT2

4 (2.8%)

1 (1.8%)

3 (3.6%)

2 (1.7%)

 pT3

138 (95.2%)

54 (98.2%)

79 (94.0%)

118 (97.5%)

 pT4

2 (1.4%)

0 (0.0%)

1 (1.2%)

1 (0.8%)

pT stage (AJCC 8th Ed.)

0.054

0.115

 pT1

12 (8.3%)

12 (21.8%)

8 (9.5%)

17 (14.0%)

 pT2

89 (61.4%)

28 (50.9%)

57 (67.9%)

62 (51.2%)

 pT3

42 (29.0%)

15 (27.3%)

18 (21.4%)

41 (33.9%)

 pT4

2 (1.4%)

0 (0.0%)

1 (1.2%)

1 (0.8%)

pN stage (AJCC 7th Ed.)

0.493

0.202

 pN0

52 (36.6%)

23 (41.8%)

28 (33.3%)

51 (42.1%)

 pN1

93 (63.4%)

32 (58.2%)

56 (66.7%)

70 (57.9%)

pN stage (AJCC 8th Ed.)

0.035

0.427

 pN0

53 (36.6%)

23 (41.8%)

28 (33.3%)

51 (42.1%)

 pN1

73 (50.3%)

18 (32.7%)

42 (50.0%)

51 (42.1%)

 pN2

19 (13.1%)

14 (25.5%)

14 (16.7%)

19 (15.7%)

Distant metastasis

0.058

0.427

 Absent

55 (37.9%)

29 (52.7%)

38 (45.2%)

48 (39.7%)

 Present

90 (62.1%)

26 (47.3%)

46 (54.8%)

73 (60.3%)

Local recurrence

0.364

0.682

 Absent

82 (56.6%)

35 (63.6%)

51 (60.7%)

70 (57.9%)

 Present

63 (43.4%)

20 (36.4%)

33 (39.3%)

51 (42.1%)

On combining the expression status of the two markers, PDACs with RUNX3-/SMAD4+ profiles showed less frequent distant metastasis (p = 0.017) and tended to be smaller in size (p = 0.092) compared to the other PDACs which showed SMAD4 loss and/or RUNX3 expression (Table 3).

Table 3: Relationship between RUNX3/SMAD4 combined status and clinicopathological features in pancreatic ductal adenocarcinoma cases

Parameters

RUNX3-/SMAD4+

Others* (RUNX3+SMAD4+, SMAD4- groups)

p-value

Sex

0.383

 Male

13 (52.0%)

107 (61.1%)

 Female

12 (48.0%)

68 (38.9%)

Age at operation (mean ± SD)

65.2 ± 8.36

64.2 ± 10.22

0.605

Tumor size

0.092

 ≤ 2 cm

5 (20.0%)

19 (10.9%)

 > 2 cm and ≤ 4 cm

17 (68.0%)

101 (57.7%)

 > 4 cm

3 (12.0%)

55 (31.4%)

Histologic grade

0.153

 WD/MD

24 (96.0%)

150 (85.7%)

 PD/UD

1 (4.0%)

25 (14.3%)

Lymphatic invasion

0.611

 Absent

15 (60.0%)

95 (54.6%)

 Present

10 (40.0%)

79 (45.4%)

Venous invasion

0.681

 Absent

17 (68.0%)

111 (63.8%)

 Present

8 (32.0%)

63 (36.2%)

Perineural invasion

1.000

 Absent

4 (16.0%)

27 (15.5%)

 Present

21 (84.0%)

147 (84.5%)

pT stage (AJCC 7th Ed.)

0.755

 pT1

0 (0.0%)

1 (0.6%)

 pT2

0 (0.0%)

5 (2.9%)

 pT3

25 (100.0%)

167 (95.4%)

 pT4

0 (0.0%)

2 (1.1%)

pT stage (AJCC 8th Ed.)

0.173

 pT1

5 (20.0%)

19 (10.9%)

 pT2

17 (68.0%)

100 (57.1%)

 pT3

3 (12.0%)

54 (30.9%)

 pT4

0 (0.0%)

2 (1.1%)

pN stage (AJCC 7th Ed.)

0.509

 pN0

11 (44.0%)

65 (37.1%)

 pN1

14 (56.0%)

110 (62.9%)

pN stage (AJCC 8th Ed.)

0.592

 pN0

11 (44.0%)

65 (37.1%)

 pN1

9 (36.0%)

82 (46.9%)

 pN2

5 (20.0%)

28 (16.0%)

Distant metastasis

0.017

 Absent

16 (64.0%)

68 (38.9%)

 Present

9 (36.0%)

107 (61.1%)

Local recurrence

0.303

 Absent

17 (68.0%)

100 (57.1%)

 Present

8 (32.0%)

75 (42.9%)

*Others: RUNX3-/SMAD4-, RUNX3+/SMAD4−, RUNX3+/SMAD4+.

Survival analysis

The univariate survival analysis results are summarized in Table 4 and Figure 2. Clinicopathological parameters significantly associated with poor overall survival included large tumor size (p = 0.032), presence of lymphatic (p = 0.005), venous (p < 0.001) or perineural invasion (p = 0.007), high pT stage (7th ed.) (p = 0.041) and high pN stage (7th ed. p < 0.001, 8th ed. p = 0.001). Similar findings were seen for progression-free survival: larger tumor size (p = 0.013), presence of lymphatic (p = 0.003), venous (p = 0.003) or perineural invasion (p = 0.007), high pT stage (7th ed. p = 0.014, 8th ed. p = 0.018), and high pN stage (7th ed. p = 0.003, 8th ed. p = 0.011) were significantly associated with reduced progression-free survival.

Table 4: Survival analysis in clinicopathological variables and immunohistochemical results

Parameters

Overall survival

Progression–free survival

Median survival time (month, 95% CI)

Number of events

p-value

Median survival time (month, 95% CI)

Number of events

p-value

Sex

0.169

0.189

 Male

19.00 (15.81–22.18)

111/126

10.00 (6.10–13.89)

113/125

 Female

17.00 (11.40–22.59)

63/84

8.00 (6.36–9.63)

67/84

Age at operation

0.376

0.715

 ≤ 65 years

19.00 (13.96–24.03)

88/108

10.00 (6.40–13.59)

91/107

 < 65 years

16.00 (12.27–19.72)

86/102

8.00 (5.67–10.32)

89/102

Tumor size

0.032

0.013

 ≤ 2cm

29.00 (19.00–38.99)

20/26

16.00 (9.82–22.17)

21/26

 > 2cm and ≤ 4cm

19.00 (15.40–22.59)

103/123

10.00 (5.67–14.32)

105/122

 > 4cm

12.00 (8.79–15.20)

51/61

5.00 (3.49–6.50)

54/61

Histologic grade

0.124

0.391

 WD/MD

19.00 (15.59–22.40)

150/182

9.00 (6.89–11.10)

157/182

 PD/UD

11.00 (7.12–14.87)

24/28

5.00 (1.60–8.39)

23/27

Lymphatic invasion

0.005

0.003

 Absent

21.00 (16.51–25.48)

91/114

15.00 (10.83–19.16)

94/113

 Present

14.00 (11.44–16.44)

83/95

6.00 (3.96–8.03)

86/95

Venous invasion

< 0.001

0.003

 Absent

23.00 (19.05–26.94)

106/134

11.00 (6.83–15.16)

111/133

 Present

13.00 (10.52–15.47)

68/75

6.00 (4.14–7.86)

69/75

Perineural invasion

0.007

0.007

 Absent

24.00 (0.00–59.25)

20/32

19.00 (2.36–35.63)

22/32

 Present

17.00 (14.66–19.34)

154/177

8.00 (6.00–9.99)

158/176

pT stage (7th Ed.)

0.041

0.014

 pT1

8.00

1/2

8.00

1/2

 pT2

*

1/5

*

1/5

 pT3

18.00 (15.62–20.37)

170/201

9.00 (7.09–10.90)

176/200

 pT4

14.00

2/2

*

2/2

pT stage (8th Ed.)

0.055

0.018

 pT1

29.00 (19.00–38.99)

20/26

16.00 (9.82–22.17)

21/26

 pT2

19.00 (15.42–22.57)

102/122

10.00 (5.69–14.30)

104/121

 pT3

12.00 (8.82–15.17)

50/60

5.00 (3.39–6.60)

53/60

 pT4

14.00

2/2

4.00

2/2

pN stage (7th Ed.)

< 0.001

0.003

 pN0

27.00 (16.02–37.97)

59/80

14.00 (7.22–20.77)

63/79

 pN1

16.00 (13.64–18.35)

115/130

8.00 (6.04–9.95)

117/130

pN stage (8th Ed.)

0.001

0.011

 pN0

27.00 (16.02–37.97)

59/80

14.00 (7.22–20.77)

63/79

 pN1

15.00 (12.62–17.37)

85/97

8.00 (6.41–9.58)

87/97

 pN2

17.00 (13.78–20.21)

30/33

9.00 (4.98–13.01)

30/33

SMAD4 status

0.015

0.044

 Loss

16.00 (13.54–18.45)

127/145

8.00 (5.95–10.04)

130/144

 Intact

25.00 (19.15–30.84)

39/55

11.00 (6.21–15.78)

42/55

RUNX3 status

0.010

0.009

 No expression

23.00 (18.51–27.48)

66/84

12.00 (5.41–18.58)

68/84

 Expression

16.00 (13.57–18.42)

104/121

8.00 (5.53–10.46)

108/120

4–group combination

0.015

0.030

 RUNX3–/SMAD4–

19.00 (14.92–23.07)

48/56

8.00 (0.67–15.32)

49/56

 RUNX3–/SMAD4+

34.00 (12.78–55.21)

16/25

29.00 (0.00–58.37)

17/25

 RUNX3+/SMAD4–

15.00 (12.74–17.25)

79/89

8.00 (5.70–10.29)

81/88

 RUNX3+/SMAD4+

20.00 (13.72–26.27)

23/30

7.00 (1.72–12.27)

25/30

2–group combination

0.008

0.009

 RUNX3–/SMAD4+

34.00 (12.78–55.21)

16/25

29.00 (0.00–58.37)

17/25

 Others**

17.00 (14.72–19.27)

150/175

8.00 (6.01–9.98)

155/174

* All cases were censored.

** RUNX3–/SMAD4–, RUNX3+/SMAD4–, RUNX3+/SMAD4+.

Survival analysis results.

Figure 2: Survival analysis results. Decreased overall survival (OS) (A) and progression-free survival (PFS) (B) are seen in PDACs with SMAD4 loss compared to those with intact SMAD4. RUNX3-expressing PDACs demonstrate decreased OS (C) and PFS (D) compared to RUNX3-negative tumors. RUNX3−/SMAD4+ PDACs show increased OS (E, G) and PFS (F, H) compared to the other PDACs. (G, H) “Other groups”: RUNX3−/SMAD4−, RUNX3+/SMAD4−, RUNX3+/SMAD4+ PDACs).

When the survival was analyzed according to the immunohistochemical stain results, loss of SMAD4 expression was significantly associated with poor overall survival (median survival 16 months versus 25 months, p = 0.015) and progression-free survival (median survival 8 months versus 11 months, p = 0.044) compared to PDACs with intact SMAD4 expression. RUNX3 expression was significantly associated with decreased overall survival (median survival 16 months versus 23 months, p = 0.010) and progression-free survival (median survival 8 months versus 12 months, p = 0.009) compared to RUNX3-negative PDACs. When the RUNX3/SMAD4 status was combined, RUNX3-/SMAD4+ tumors were associated with remarkably longer overall survival (median 34 months versus 17 months, p = 0.008) and progression-free survivals (median 29 months versus 8 months, p = 0.009) compared to other groups. Interestingly, among the PDACs with no RUNX3 expression, those with SMAD4 loss demonstrated similarly decreased overall and progression-free survivals as RUNX3-expressing PDACs.

After adjusting for sex and age, RUNX3-/SMAD4+ status remained a significant independent predictive factor for favorable overall [p = 0.025, HR 1.842 (95% CI 1.079–3.143)] and progression-free survival [p = 0.020, HR 1.850 (95% CI 1.100–3.113)] on multivariate analysis, in addition to tumor size (Table 5).

Table 5: Multivariate analysis for overall survival and progression-free survival

Parameters

Overall survival

Progression-free survival

Hazard ratio (95% CI)

p-value

Hazard ratio (95% CI)

p-value

Variables adjusted for age and sex

RUNX3−/SMAD4+ vs. others*

1.842 (1.079–3.143)

0.025

1.850 (1.100–3.113)

0.020

Histologic grade

1.517 (0.961–2.393)

0.073

1.163 (0.730–1.852)

0.525

Tumor size

≤ 2 cm

1

Reference

1

Reference

> 2 cm and ≤ 4 cm

1.511 (0.912–2.503)

0.109

1.298 (0.787–2.142)

0.307

> 4 cm

2.251 (1.287–3.935)

0.004

2.191 (1.248–3.847)

0.006

Lymphatic invasion

1.330 (0.962–1.839)

0.085

1.492 (1.101–2.023)

0.010

Venous invasion

1.547 (1.100–2.176)

0.012

1.267 (0.896–1.792)

0.180

Perineural invasion

1.546 (0.935–2.557)

0.089

1.565 (0.982–2.494)

0.059

pT stage (7th Ed.)

8.008 (1.071–59.866)

0.043

10.211 (1.380–75.567)

0.023

pN stage (7th Ed.)

1.178 (0.837–1.658)

0.348

1.057 (0.758–1.474)

0.745

* RUNX3-/SMAD4-, RUNX3+/SMAD4-, RUNX3+/SMAD4+.

Comparison of immunohistochemical status between primary and metastatic lesions

Of the 210 patients studied, 121 (57.6%) subsequently developed distant metastases. Biopsies or resected tissues from metastatic sites were available for 14/121 patients and the whole tissue sections of the 14 metastatic tumors were subjected to SMAD4 and RUNX3 immunohistochemistry. The liver was the most common site of distant metastasis (n = 9) (Table 6).

Table 6: Comparison of SMAD4 and RUNX3 labeling status between primary and metastatic tumors (n = 14)

Case#

Status of primary site

Metastatic sites

Status of metastatic site

1

RUNX3+/SMAD4−

Liver

RUNX3−/SMAD4−

2

RUNX3+/SMAD4−

Liver

RUNX3−/SMAD4−

3

RUNX3+/SMAD4

Liver

RUNX3+/SMAD4−

4

RUNX3+/SMAD4−

Cervical lymph node

RUNX3−/SMAD4−

5

RUNX3+/SMAD4−

Lung

RUNX3−/SMAD4−

6

RUNX3+/SMAD4−

Liver

RUNX3+/SMAD4+

7

RUNX3+/SMAD4−

Liver

RUNX3−/SMAD4−

8

RUNX3-/SMAD4

Liver

RUNX3−/SMAD4−

9

RUNX3+/SMAD4+

Liver

RUNX3−/SMAD4−

10

RUNX3-/SMAD4

Liver

RUNX3−/SMAD4−

11

RUNX3-/SMAD4

Liver

RUNX3−/SMAD4−

12

RUNX3+/SMAD4+

Bone

RUNX3−/SMAD4−

13

RUNX3+/SMAD4−

Lung

RUNX3−/SMAD4−

14

RUNX3-/SMAD4

Colon

RUNX3−/SMAD4−

Identical RUNX3/SMAD4 profiles were seen in the primary and metastatic tumors of 5/14 (35.7%) cases. While 11/14 (78.6%) of the cases showed concordant SMAD4 expression status between the matched primary and metastatic tumors, only 6/14 (42.9%) cases showed concordant RUNX3 expression status. Interestingly, out of the 14 matched cases, the RUNX3-/SMAD4+ profile was seen in none of the primary PDACs and also in none of their subsequent metastatic tumors. In addition, only 1/14 (7.1%) of the metastatic tumors had intact SMAD4 expression, and all 8 (100%) cases with discordant RUNX3 status were RUNX3+ in the primary tumor and RUNX3-negative in the matched metastatic tumors.

DISCUSSION

The purpose of this study was to evaluate the expression status of SMAD4 and RUNX3 in PDACs, and to explore the utility of the SMAD4/RUNX3 marker combination assay in predicting PDAC behavior. We demonstrate in this retrospective study of 210 PDACs that the combination of intact SMAD4 and RUNX3 negativity may help to identify a subset of PDAC patients with favorable prognosis.

SMAD4 (or DPC4) is a well-known tumor suppressor gene that is inactivated in the late stages of pancreatic carcinogenesis: SMAD4 inactivation has been reported in ~50% of PDACs and in ~30% of high-grade pancreatic intraepithelial neoplasia (PanIN-3) [912]. Although the majority of clinicopathological studies, including our present study, have demonstrated associations between SMAD4 loss in PDACs and poor prognoses, there are also reports showing no significant correlations with survival, or even evidence for improved survival in PDACs with SMAD4 loss [1317]. Loss of SMAD4 protein expression has been demonstrated to be a sensitive and specific surrogate marker for SMAD4 alteration, independent of whether SMAD4 inactivation occurred by homozygous deletions or by loss of heterozygosity, and the presence of intact SMAD4 protein expression corresponds to tumors with at least one wild-type allele (i.e. SMAD4(+/+) or SMAD4(+/)) [11, 18]. Therefore, PDACs with intact SMAD4 protein expression would comprise a mixed population of SMAD4(+/+) and SMAD4(+/) PDACs, and hence the discrepancies in the clinicopathological studies on the prognostic significance of SMAD4 immunohistochemistry are not surprising. In addition, PDACs with SMAD4 loss tended to show more frequent distant metastasis on follow-up compared to SMAD4-intact tumors, while there was no difference in the frequency of local progression according to SMAD4 status, supporting the previous literature that suggested correlations between SMAD4 status and disease progression patterns in PDAC [19, 20].

The role of RUNX3 in cancer is still not well-established; it has been described to function as a tumor suppressor in most malignancies, including cancers of the breast, stomach, liver, lung and prostate, [2125] but tumor promoting roles of RUNX3 have been suggested for some tumors such as ovarian cancers, melanomas and PDACs [8, 2628]. An association between RUNX3 protein expression and decreased overall survival has also been demonstrated in PDAC patients, although in a small number of cases [8]. A recent genetically engineered mouse model study by Whittle et al. demonstrated an interesting relationship between SMAD4 and RUNX3 status and the biological behavior (metastasis versus primary tumor growth) of PDACs according to SMAD4/RUNX3 status [7, 8, 29]. In brief, KPC (SMAD4(+/+)) and KPDDC (SMAD4(/)) tumors showed increased RUNX3 expression levels and a high metastatic disease burden, and the high expression of RUNX3 was associated with increased metastatic potential of PDACs independent of epithelial-mesenchymal transition [8, 30]. On the other hand, SMAD4 haploinsufficient KPDC mice (SMAD4(+/)) lacked RUNX3 expression, had a significantly lower metastatic burden, but showed an increased proliferative activity of the primary tumor compared to KPC tumors. We did not find differences in the proliferative activity (Ki-67 labeling index) of PDACs according to SMAD4/RUNX3 status in this study (data not shown), but we did find that PDACs with RUNX3 expression and/or SMAD4 loss were more likely to metastasize to distant sites and follow an aggressive clinical course compared to PDACs that did not show these immunophenotypes. Therefore, combining the SMAD4 and RUNX3 protein expression status would provide a more accurate prognostic marker compared to SMAD4 status alone.

It is also interesting that when the immunohistochemical profiles of the primary and metastatic tumors were compared in the separate analysis of 14 primary versus matched metastatic PDACs, SMAD4 labeling status was concordant in the majority of the cases, while the RUNX3 expression status were more frequently discordant. All primary tumors showed RUNX3 expression and/or SMAD4 loss; RUNX3-/SMAD4+ status was not seen in any of the primary or metastatic tumors in this analysis. In addition, all RUNX3-discordant cases showed conversion from RUNX3-positivity in the primary tumor to RUNX3-negativity in the matched metastatic tumors. Attenuation of RUNX3 levels once the metastatic niche is established, switching the metastatic tumor to the locally proliferative state, would be an attractive explanation for this observation; however, experimental evidence would be required to support this.

This is to our knowledge the first and the largest single institutional clinicopathological study demonstrating RUNX3/SMAD4 combination immunohistochemistry to be a useful predictor of both overall and disease-free survivals in PDAC patients. Although further validation in independent cohorts would be required, application of RUNX3/SMAD4 immunohistochemistry on biopsies or resected PDAC tissues may not only be a prognostic marker but also help guide treatment plans in PDAC patients; RUNX3−/SMAD4+ PDACs are less likely to metastasize to distant sites and may therefore be candidates for more aggressive local surgery and radiotherapy, whereas PDACs with RUNX3 expression and/or SMAD4 loss profiles may benefit more from systemic chemotherapy.

MATERIALS AND METHODS

Patient selection and clinicopathological analysis

This retrospective analysis was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB #B-1611-369-302). The study subjects comprised 210 consecutive cases of PDACs resected between June 2003 and January 2013 at Seoul National University Bundang Hospital, Seongnam, South Korea. Clinicopathological data were retrieved by reviewing electronic medical records, pathology reports and glass slides, and included patient sex, initial treatment, age at operation, tumor size, histologic differentiation, serum tumor marker levels (CEA, CA19–9), and pathological T and N stages according to American Joint Committee on Cancer (AJCC) 7th edition and the new 8th edition. Follow up data was also retrieved from the electronic medical records, including the status at last follow up, and occurrence of local recurrence and/or distant metastasis during the follow up period. The dates of local recurrence and distant metastasis were defined as when new lesions appeared on imaging studies or when tumor marker levels were elevated. The date of disease progression was defined as when the event has occurred postoperatively such as local recurrence, distant metastasis or death.

Immunohistochemistry

Two mm-core tissue microarrays were constructed from 210 PDACs and their matched non-neoplastic pancreatic tissues (Superbiochips Laboratories, Seoul, Korea), and 4 μm-thick tissue sections obtained from the microarray blocks were subjected to immunohistochemical staining for SMAD4 (1:100, rabbit monoclonal, Abcam, Cambridge, UK) and RUNX3 (1:100, rabbit monoclonal, Cell signaling technology, Denver, USA). In brief, tissue sections were deparaffinized in xylene, rehydrated in graded alcohol, and antigen retrieval was performed using citrate buffer (pH 6.0). Incubation with primary antibodies was performed for 1 hour, and then with secondary antibodies (EnVision Detection System, Dako) for 30 minutes. Counterstaining was performed using Mayer’s hematoxylin.

The labeled slides were interpreted by 2 pathologists (YL and HK). SMAD4 was expressed in the cytoplasm and nuclei of normal pancreatic duct epithelia, acinar cells, lymphocytes, stromal fibroblasts and endocrine cells, and served as positive controls. Expression in < 20% of tumor cell nuclei was defined as SMAD4 loss (“SMAD4-”). RUNX3 was not expressed in normal pancreatic ductal epithelial cells or acinar cells, but was expressed in the lymphocytes. RUNX3 expression status was evaluated in terms of both intensity and distribution of staining. The staining intensity was defined as follows: “0”, no expression; “1”, weaker intensity compared to staining of lymphocytes; “2”, strong intensity similar to that seen in lymphocytes. For distribution, the percentage of positively stained tumor cell nuclei was estimated in 5% increments. The staining distribution was relatively homogeneous throughout the tumor. The presence of weak or strong RUNX3 positivity in 5% or more of tumor cells were regarded as positive (“RUNX3+”) for subsequent analysis.

For cases where both primary pancreatic tumors and metastatic tumors were available for examination, whole tissue sections were subjected to the same immunohistochemical stains for RUNX3 and SMAD4, and the expression status of both markers were compared between the primary and matched metastatic tumors. These cases were from the same cohort of 210 PDACs.

Statistical analysis

All statistical analyses were performed using SPSS 19.0K (SPSS Korea, Seoul, South Korea). Chi-square tests, Fisher exact tests and Student t-tests were performed as deemed appropriate. Univariate survival analyses for overall and progression-free survivals were performed by the Kaplan-Meier method and log-rank test. The Cox regression models were used for multivariate analysis. Statistical significance was defined as p < 0.05.

CONFLICTS OF INTEREST

The authors declare no potential conflicts of interest.

FUNDING

This study was supported by grant number 02-2016-008 from the SNUBH Research Fund, and the Basic Science Research Program through NRF funded by the Ministry of Education (NRF-2016R1D1A1A09919042) to HK.

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