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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 809-813
Low expression of DUSP4 expression predicts unfavorable prognosis in gallbladder adenocarcinoma


1 Department of Pathology, Hanyang University College of Medicine, Seoul, Republic of Korea
2 Department of Pathology, Korea University College of Medicine, Seoul, Republic of Korea
3 Department of Surgery, Hanyang University College of Medicine, Seoul, Republic of Korea
4 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea

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Date of Submission06-Apr-2021
Date of Decision30-May-2021
Date of Acceptance03-Jun-2021
Date of Web Publication21-Oct-2022
 

   Abstract 


Background: Dual specificity phosphatase 4 (DUSP4), which regulates the mitogen activated protein kinases, has emerged as a tumor suppressor gene in several human malignancies. Aims and Objectives: In this study, we investigated the clinicopathologic significance and the prognostic role of DUSP4 in gallbladder adenocarcinoma. Materials and methods: DUSP4 expression was evaluated immunohistochemically in tissue microarray from 110 gallbladder adenocarcinoma samples and scored by H score system. The cut off (H score <170) was determined by ROC curve analysis. Results: Low expression of DUSP4 expression was observed in 57 (51.8%) out of 110 gallbladder adenocarcinoma samples. Low expression of DUSP4 expression was significantly associated with high histologic grade (P = 0.017), high pT stage (P = 0.002) and high AJCC stage (P = 0.007). Kaplan Meier survival curves revealed that patients with low expression of DUSP4 expression had significantly worse cancer specific survival (P = 0.024, log rank test). However, there was no significant association between DUSP4 expression and recurrence free survival. Conclusions: In conclusion, gallbladder adenocarcinoma with low expression of DUSP4 expression was associated with adverse clinicopathologic characteristics and poor patient outcome.patient outcome.

Keywords: Dual-specificity phosphatase 4, gallbladder adenocarcinoma, immunohistochemistry, prognosis

How to cite this article:
Jee S, Sim J, Bang S, Park H, Myung J, Paik SS, Choi D, Kim Y, Kim H. Low expression of DUSP4 expression predicts unfavorable prognosis in gallbladder adenocarcinoma. Indian J Pathol Microbiol 2022;65:809-13

How to cite this URL:
Jee S, Sim J, Bang S, Park H, Myung J, Paik SS, Choi D, Kim Y, Kim H. Low expression of DUSP4 expression predicts unfavorable prognosis in gallbladder adenocarcinoma. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Nov 30];65:809-13. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/809/359359





   Background Top


Gallbladder adenocarcinoma (GBA) accounts for 1.2% of global cancer diagnoses and is the most common malignancy of biliary tract.[1] The incidence of GBA shows characteristic geographical variations with quite low incidences in most Western countries.[2] In contrast, high incidence and high mortality rates were reported in South Asia, South America and East Asian countries including Japan, China and Korea.[3] Although surgical resection remains to be the front-line treatment, many of the GBAs are not resectable at the time of presentation and this contributes to the dismal prognosis. The reported 5-year survival of GBA is 18% and the median survival is 12-14 months.[1] While many candidate biomarkers have been investigated for prediction of GBA's prognosis and targeted therapy, these remain still controversial.[3] Therefore, finding a novel biomarker of GBA is required to improve clinical management and increase treatment options.

Dual-specificity phosphatase 4 (DUSP4), also known as MAPK phosphatase 2 (MKP2), belongs to the DUSP family.[4] DUSP family specifically dephosphorylates the mitogen-activated protein kinase (MAPK), ERK1/2, p38 and JNK and functions as negative regulators of MAPK activities.[5] DUSP4 plays an essential role in regulation of cell proliferation, differentiation and apoptosis via interactions with the MAPK signaling pathway.[5] Therefore, aberrant DUSP4 expression would impact the cellular phenotypes leading to tumorigenesis and the relationship between DUSP4 expression and tumor progression has been reported.[6]

Down regulation or loss of DUSP4 expression has been reported in several types of cancers including gastric cancer, colorectal cancer, pancreatic cancer, lung cancer and breast cancer.[7],[8],[9],[10],[11] Interestingly, DUSP4-related drug resistance also has been reported in gastric cancer, colorectal cancer and breast cancer.[12],[13],[14] The involvement of DUSP4 in resistance to cancer therapy has especially gained prominence, as it implies that DUSP4 could potentially be a new target of anticancer therapy. However, expression of DUSP4 and its clinicopathologic significance in GBA has not yet been identified.

We investigated the immunohistochemical expression of DUSP4 in 110 cases of GBA and analyzed the correlation of its expression with various clinicopathologic factors to determine the prognostic significance of DUSP4.


   Materials and Methods Top


Patients

From January 1991 to December 2018, 110 patients underwent curative surgery for primary GBA at Hanyang University Hospital. Patients who had received neoadjuvant treatment, or those who had recurrent GBA were excluded. To determine the clinical characteristics, medical records were reviewed for the following parameters: age, sex, survival status, recurrence status and follow-up interval. Two surgical pathologists (S. J. and Y. K.) reviewed the pathology reports and hematoxylin and eosin-stained slides to determine the pathologic parameters and to confirm the diagnosis. Pathologic parameters including TNM stage (8th AJCC) were determined according to a protocol for examining specimens from patients with carcinoma of the gallbladder (the Collage of American Pathologists).[15] The pathologic characteristics included gross type, tumor location, histologic grade, lymphovascular invasion, perineural invasion, and TNM stage. This study was approved by the Institutional Review Board of the Hanyang University Hospital (HYUH 2018-08-031-002), and the requirement for informed consent was waived.

Tissue microarray construction

Manual tissue microarrayer (Unitma, Seoul, Korea) was used for tissue microarray construction from archival formalin-fixed, paraffin-embedded tissue blocks. We marked a representative tumor lesion on H and E-stained sections by light microscopy. Tissue cylinders with 2 mm diameter were punched from a previously marked area of tumor lesion on donor block and transferred to the recipient block (Unitma, Seoul, Korea).

Immunohistochemical study and interpretation

The immunohistochemical study for DUSP4 expression was performed with 4-μm-thick sections from TMA blocks using the Ventana Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer's protocol. The rabbit monoclonal anti-DUSP4 (1:200, ab72593, Abcam, Cambridge, UK) antibody was used for immunohistochemical staining. DUSP4 expression was scored according to the fraction of stained cells at each intensity. The staining intensity was scored within a scale ranging from 0 to 3 and divided into 4 categories as follows: 0 (negative), 1 (weak staining), 2 (intermediate staining), and 3 (strong staining). The DUSP4 H-score was defined as a continuous variable with a scale ranging from 0-300 and was calculated using the following formula: 1× (percentage of weakly stained cells) +2× (percentage of moderately stained cells staining) +3× (percentage of strongly stained cells. We performed ROC curve analysis for survival endpoints to determine the cut-off value for (low or high) DUSP4 expression. An H-score <170 was defined as low DUSP4 expression.

Statistical analysis

Statistical analysis was performed using a SPSS software version 21 (IBM Corp., Armonk, USA). The Chi-square test was used to evaluate the association between DUSP4 expression and other clinicopathologic parameters including sex, age, histologic grade, lymphovascular invasion, perineural invasion and TNM stage. Cancer-specific survival (CSS) was defined as the time interval between the date of curative surgery and the date of death due to GBA. Recurrence-free survival (RFS) was defined as the time interval between the date curative surgery and the date of recurrence. Survival analysis was performed using Kaplan-Meier survival curves and the log-rank test. Two-tailed P values <0.05 were considered statistically significant.


   Results Top


Baseline characteristics of the cohort

The clinicopathologic characteristics of patients are summarized in [Table 1]. The mean follow-up period was 48 months (range 1–120), and the mean age at surgery was 65.2 years (range 28–90). 55 (50.0%) patients were women with a male-to-female ratio of 1:1. Pathologic evaluation revealed that 27 cases (24.5%) were histologic grade 1, 60 (54.5%) were grade 2, and 23 (20.9%) were grade 3. According to the 8th AJCC staging system, 19 (17.3%) were stage I, 37 (33.6%) were stage II, 49 (45.4%) were stage III, and 5 (4.5%) were stage IV.
Table 1: Baseline characteristics of the cohort

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Correlation between DUSP4 expression and clinicopathologic features

Low expression of DUSP4 expression was observed in 57 (51.8%) patients. Representative microscopic photos are shown in [Figure 1]. The associations between DUSP4 expression and clinicopathologic factors are summarized in [Table 2]. The low expression of DUSP4 expression was significantly correlated with high histologic grade (P = 0.017, Chi-square test), pathologic T (pT) stage (P = 0.002, Chi-square test) and AJCC stage (P = 0.007, Chi-square test). These results demonstrate that low expression of DUSP4 expression is associated with adverse pathological features in GBA.
Figure 1: Representative microphotos of gallbladder adenocarcinoma (GBA) with high and low DUSP4 expression. (a and b) Hematoxylin and eosin staining of GBA tissues. (c and d) low magnification microphotos of high DUSP4 expression and low DUSP4 expression. (e and f) high magnification microphotos of high DUSP4 expression and low DUSP4 expression. (c and e, corresponding to panel a; d and f, corresponding to panel b). Original magnification, a and b, ×200; c and d, ×100; e and f, ×400

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Table 2: Correlation between DUSP4 expression and clinicopathologic factor

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Prognostic significance of DUSP4 expression

Kaplan-Meier survival curves revealed that patients with low expression of DUSP4 expression had a poor prognosis in cancer-specific survival (P = 0.024, log-rank test) [Figure 2]a. There was a tendency of patients with low expression of DUSP4 expression to show a poor outcome in cancer-specific survival, but this result was not statistically significant (P = 0.105, log-rank test) [Figure 2]b. Univariate survival analyses revealed as predictors of poor CSS in low expression of DUSP4 expression (P = 0.027), lymphovascular invasion (P = 0.004), perineural invasion (P = 0.001), pT stage (P < 0.001), pN stage (P = 0.004) and AJCC stage (P < 0.001) as predictors of poor cancer-specific survival (CSS) in GBA patients [Table 3]. Similarly, lymphovascular invasion (P < 0.001), perineural invasion (P < 0.001), pT stage (P < 0.001), pN stage (P < 0.001) and AJCC stage (P < 0.001) were negatively correlated with recurrence-free survival (RFS) [Table 3]. However, DUSP4 expression was not an independent prognostic factor for cancer-specific survival or recurrence-free survival in the multivariate analysis with the Cox proportional hazards model (P = 0.179 and P = 0.656, respectively) [Table 4].
Figure 2: Kaplan–Meier survival curves of gallbladder adenocarcinoma patients stratified by DUSP4 expression. Cancer-specific survival (a) and recurrence-free survival (b) according to DUSP4 expression

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Table 3: Univariate Cox regression analysis of prognostic factors for cancer-specific survival and recurrence-free survival in gallbladder adenocarcinoma

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Table 4: Multivariate Cox regression analysis of prognostic factors for cancer-specific survival and recurrence-free survival in gallbladder adenocarcinoma

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   Discussion Top


The MAPKs are key regulators of cell proliferation, survival and apoptosis in physiologic and pathologic processes.[5] The MKPs, also known as DUSPs, act as major negative regulators of MAPK activities.[16] Aberrant expression of DUSP has been observed in various human malignancies, emphasizing the importance of DUSP's role in carcinogenesis.[16] Down regulation or loss of DUSP4 expression has been observed in several types of cancers including gastric cancer, colorectal cancer, pancreatic cancer, lung cancer and breast cancer.[7],[8],[9],[10],[11] In gastric cancer, DUSP4 overexpression inhibited tumor cell viability and invasiveness, and induced cell cycle arrest and apoptosis.[7] Ichimanda M et al.[8] reported downregulation of DUSP4 enhances tumor cell proliferation and invasiveness in colorectal cancer. Hijiya N et al.[9] analyzed the genomic profiles of noninvasive carcinoma and invasive carcinoma of pancreas and highlighted DUSP4 as a novel invasion suppressor. In lung cancer, DUSP4 has been reported as a growth inhibitor in EGFR-mutant lung adenocarcinoma.[10] Armes JE et al.[11] revealed frequent DUSP4 protein loss in breast carcinomas and suggested DUSP4 as a candidate tumor-suppressor gene contributing to the chromosome arm 8p loss in breast carcinogenesis.

Recent studies reporting a DUSP-mediated resistance to cancer therapies have led to a raised interest for the use of DUSP4 as a potential target of anticancer therapy.[5] Several studies have attempted to identify specific inhibitors targeting DUSPs which have anti-tumor properties.[17],[18],[19] Sanguinarine, benzo[c] phenanthridine alkaloid, was identified as a selective, cell-active inhibitor of MKP-1 in HeLa cancer cell line.[17] Another MKP-1 inhibitor, triptolide, also has been identified to be an enhancer of cisplatin-mediated apoptosis in ovarian cancer cells.[18] Vogt and Lazo identified NSC95397, a compound molecule which inhibits both MKP-1 and MKP-3, and restored paclitaxel sensitivity in breast cancer cells.[19] In Burkitt's lymphoma cells, transcriptional silencing of MKP7 eliminated the negative regulation of JNK activity enhancing the sensitivity of tumor cells to chemotherapeutic agents including doxorubicin, sorbitol and cisplatin.[20] Although only studies on anticancer process of MKP-1, MKP-3 and MKP-7 inhibitors have been reported, the use of specific phosphatase inhibitors targeting the other MKPs including DUSP4 (MKP-2) may provide novel therapeutic strategies against cancers.[5]

In this study, we observed low expression of DUSP4 expression in 51.8% of human GBA tissues and showed that low expression of DUSP4 expression was associated with aggressive phenotypes, including high histologic grade, high pT stage, high AJCC stage and worse cancer-specific survival. Although this is the only study in GBA determining the prognostic role of DUSP4 expression, there are several limitations. Firstly, this study was retrospective and selection bias was not completely excluded. Secondly, the cohort was obtained in a single medical center with a limited number of patients. Thirdly, functional studies revealing tumor suppressive role of DUSP4 has not been performed and the detailed molecular mechanism was not explained. Further studies with larger cohort are warranted to identify the tumor suppressive function of DUSP4 in GBA.

In conclusion, we investigated the clinicopathologic significance of DUSP4 expression in 110 GBA patients. Low expression of DUSP4 expression was observed in 51.8% of GBA tissues and was significantly associated with aggressive clinicopathologic features and poor patient outcomes in GBA patients.

Declaration of patient consent

This study was approved by the Institutional Review Board of the Hanyang University Hospital (HYUH 2018-08-031-002), and the requirement for informed consent was waived.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Cushman SM, Jiang C, Hatch AJ, Shterev I, Sibley AB, Niedzwiecki D, et al. Gene expression markers of efficacy and resistance to cetuximab treatment in metastatic colorectal cancer: Results from CALGB 80203 (Alliance). Clin Cancer Res 2015;21:1078-86.  Back to cited text no. 13
    
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Keyse SM. Dual-specificity MAP kinase phosphatases (MKPs) and cancer. Cancer Metastasis Rev 2008;27:253-61.  Back to cited text no. 16
    
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Vogt A, Tamewitz A, Skoko J, Sikorski RP, Giuliano KA, Lazo JS. The benzo[c] phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1. J Biol Chem 2005;280:19078-86.  Back to cited text no. 17
    
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Wang J, Zhou JY, Zhang L, Wu GS. Involvement of MKP-1 and Bcl-2 in acquired cisplatin resistance in ovarian cancer cells. Cell Cycle (Georgetown, Tex.) 2009;8:3191-8.  Back to cited text no. 18
    
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Vogt A, McDonald PR, Tamewitz A, Sikorski RP, Wipf P, Skoko JJ 3rd, et al. A cell-active inhibitor of mitogen-activated protein kinase phosphatases restores paclitaxel-induced apoptosis in dexamethasone-protected cancer cells. Mol Cancer Ther 2008;7:330-40.  Back to cited text no. 19
    
20.
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Correspondence Address:
Hyunsung Kim
Department of Pathology, Hanyang University College of Medicine, 222 Wangsimni ro, Seongdong gu, Seoul 04763
Republic of Korea
Yeseul Kim
Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, 05505
Republic of Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_352_21

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