Significance of CD133 expression in esophageal squamous cell carcinoma

  • Hiroshi Okamoto1, 2Email author,

    Affiliated with

    • Fumiyoshi Fujishima2,

      Affiliated with

      • Yasuhiro Nakamura3,

        Affiliated with

        • Masashi Zuguchi1,

          Affiliated with

          • Yohei Ozawa1,

            Affiliated with

            • Yayoi Takahashi2,

              Affiliated with

              • Go Miyata1,

                Affiliated with

                • Takashi Kamei1,

                  Affiliated with

                  • Toru Nakano1,

                    Affiliated with

                    • Yusuke Taniyama1,

                      Affiliated with

                      • Jin Teshima1,

                        Affiliated with

                        • Mika Watanabe2,

                          Affiliated with

                          • Akira Sato1,

                            Affiliated with

                            • Noriaki Ohuchi1 and

                              Affiliated with

                              • Hironobu Sasano2, 3

                                Affiliated with

                                World Journal of Surgical Oncology201311:51

                                DOI: 10.1186/1477-7819-11-51

                                Received: 23 August 2012

                                Accepted: 12 February 2013

                                Published: 1 March 2013

                                Abstract

                                Background

                                CD133 was recently reported to be a cancer stem cell marker and a prognostic marker for several tumors. However, few studies have investigated CD133 expression in esophageal squamous cell carcinoma (ESCC). Therefore, we examined whether CD133 could serve as a prognostic marker of ESCC and investigated the correlation between CD133 expression and the clinicopathological findings of ESCC patients and several markers.

                                Methods

                                We studied 86 ESCC patients who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005. We analyzed tissue specimens by immunohistochemical staining for CD133, p53, p16, p27, murine double minute 2 (MDM2), Ki-67, and epidermal growth factor receptor (EGFR).

                                Results

                                Pathological tumor depth and tumor stage were significantly more advanced among CD133-negative patients than among CD133-positive patients. A log-rank test showed that CD133 immunoreactivity was significantly correlated with the overall survival of the patients (P = 0.049). However, multivariate analysis showed that it was not significantly correlated (P = 0.078). Moreover, CD133 was significantly positively correlated with p27 immunoreactivity (P = 0.0013) and tended to be positively correlated with p16 immunoreactivity (P = 0.057). In addition, p16 immunoreactivity was correlated with smoking history (P = 0.018), pathological lymph node status (P = 0.033), and lymphatic invasion (P = 0.018).

                                Conclusions

                                This study indicated that CD133 immunoreactivity is a good predictor of prognosis in ESCC patients. In addition, CD133 may play a role in the regulation of tumor cell cycle through p27 and p16 in ESCC. At present, it thus remains controversial whether CD133 expression is a valid prognostic marker for ESCC. To elucidate this relationship, further investigations are required.

                                Keywords

                                AC133 Esophagus Prominin-1 p16 p27 Stem cell marker

                                Background

                                Prognosis or clinical outcome of esophageal squamous cell carcinoma (ESCC) has markedly improved over the last several decades, owing to advancements in medical treatment. However, in Japan, 11,867 people succumbed to this disease in 2010, and esophageal cancer was the seventh most common cause of cancer mortality in men (3.4% of the total cancer deaths in Japan)[1]. Various prognostic markers have recently been evaluated, including the stem cell marker CD133 (Prominin-1), which was reported to be a cancer stem cell marker for cancers of the brain[2], colon[3, 4], prostate[5], liver[6, 7], lung[8], kidney[9], ovaries[10], and skin[11, 12]. It was also reported to be a marker of poor prognosis for cancers of the brain and spinal cord[13], colon[14], rectum[15], pancreas[16], breast[17], and stomach[18]. In addition, a potent cytotoxic drug, monomethyl auristatin F, which acts as an anti-CD133 antibody-drug conjugate for hepatocellular and gastric cancer cells, may be utilized to treat CD133-positive tumors[19]. To date, there have been limited studies of CD133 in esophageal cancer, and thus, the significance of CD133 in this form of cancer remains unclear. Therefore, we examined whether CD133 could serve as a prognostic marker of ESCC. In addition, we explored the correlation between CD133 expression and the clinicopathological findings of ESCC patients and the correlation between CD133 expression and the immunolocalization of several markers, such as p53, p16, p27, murine double minute 2 (MDM2), Ki-67, and epidermal growth factor receptor (EGFR), which are known as prognostic markers or tumor proliferation factors in ESCC[2027].

                                Methods

                                Patients and tissue samples

                                A total of 86 consecutive ESCC patients, who underwent curative surgery without neoadjuvant treatment at Tohoku University Hospital (Sendai, Japan) between January 2000 and December 2005, were selected. All patients underwent thoracoscopic esophagectomy with two- or three-field node dissection, except for four patients who underwent pharyngo-laryngo-esophagectomy with one-field node dissection, six patients who underwent transhiatal esophagectomy with one-field node dissection, and eight patients who underwent esophagectomy by right thoracotomy.

                                The resected specimens and lymph nodes were fixed in 10% formalin, and representative sections were embedded in paraffin wax. The sections were histologically examined according to the Union for International Cancer Control (UICC) TNM (tumor, node, metastasis) classification (7th edition) system[28]. Patient survival time was determined from the date of surgery until death, recurrence, or the last follow-up examination. This study was approved by the ethical committee of the Tohoku University Hospital (Accession number 2011–596).

                                Immunohistochemical staining and evaluation

                                Serial sections (4 μm thick), including the deepest area of the tumors, were deparaffinized in xylene, rehydrated in graded alcohol, and immersed in 3.0% hydrogen peroxide in methanol for 10 min at room temperature (RT) to inhibit endogenous peroxidase activity. For antigen retrieval, the slides for p53 were heated by microwave irradiation at 95°C for 15 min in 0.01 M citrate buffer (pH 6.0). The slides for p16, p27, MDM2, and Ki-67 were heated by autoclave at 121°C for 5 min in 0.01 M citrate buffer (pH 6.0). The slides for CD133 were autoclaved at 121°C for 5 min in Histofine antigen retrieval solution (pH 9.0, Nichirei Biosciences Inc., Tokyo, Japan). The slides for EGFR were incubated in 0.05% protease in Tris–HCl buffer (pH 7.6) at 37°C for 10 min. After washing three times for 5 min each in phosphate-buffered saline (PBS), the slides were incubated in 1% normal rabbit serum for 30 min at RT to reduce nonspecific antibody binding and were subsequently incubated at 4°C overnight with mouse monoclonal antibody against p53 (DO-7, Leica Microsystems, Bannockburn, IL, USA, diluted 1/100), p16 (G175-1239, BD Biosciences, Franklin Lakes. NJ, USA, diluted 1/100), p27 (SX53G8, Dako, Glostrup, Denmark, diluted 1/800), MDM2 (SMP14, Santa Cruz Biotechnology Inc., CA, USA, diluted 1/1000), Ki-67 (MIB-1, Dako, diluted 1/300), EGFR (31G7, Nichirei Biosciences Inc., dilution unknown, product code 413701), CD133 (AC133, Miltenyi Biotec, Auburn, CA, USA, diluted 1/10). The following day, the sections were washed three times for 5 min each in PBS, incubated with biotinylated anti-mouse immunoglobulin (Nichirei Biosciences Inc.), washed three times for 5 min each in PBS, and incubated with peroxidase-labeled streptavidin (Nichirei Biosciences Inc.) for 30 min at RT. The immunohistochemical signal was visualized with 3,3-diaminobenzidine, and the slides were counterstained with Mayer’s hematoxylin, dehydrated in graded alcohol, and cleared in xylene. For CD133, omission of the primary antibody and substitution by nonspecific immunoglobulin (Mouse IgG1, Dako) at the same concentration were used as negative and isotype controls, respectively.

                                The sections were examined by two independent observers (HO and FF) who were blinded to patients’ clinical information. The proportion of positive nuclei in more than 1,000 tumor cells of more than three fields under a ×400 magnification microscope (Leica DM LB2) at the deepest area of each tumor was calculated for p53, p27, MDM2, and Ki-67. The proportion of nuclei and cytoplasm of tumor cells positive for p16 was evaluated. The proportion of membranes of tumor cells positive for EGFR was evaluated. The cut-off values for abnormal expression were as follows: p53, ≥10%[21]; p16, ≤5%[26]; p27, ≥10%[25]; MDM2, ≥20%[29]; and Ki-67, ≥30%[27]. An immunoreactive score (IRS) was used for the scoring of EGFR. The IRS is obtained by multiplying the intensity score (0, no staining; 1, faint staining; 2, moderate staining; 3, strong staining) by the extent score (0, none; 1, <10%; 2, 10 to 50%; 3, >50 to 80%; 4, >80%) and ranges from 1 to 12. An IRS of ≥6 was defined as positive for EGFR expression[20]. When evaluating CD133, the tumors were defined as positive and negative when >1% and ≤1% of the membranes and cytoplasm of all tumor cells were immunostained, respectively[13, 30].

                                Statistical analyses

                                Statistical analyses were performed using JMP Pro Version 9.0.2 (SAS Institute Inc., Cary, NC, USA). The correlation of factors was evaluated by the chi-square test, Fisher’s exact test, or Wilcoxon test, as appropriate. Survival curves were determined by the Kaplan-Meier method, and differences in survival between groups were compared by the log-rank test. The Cox proportional hazard model was used for multivariate analysis. A P value of <0.05 was considered statistically significant.

                                Results

                                Correlation between CD133 and clinicopathological findings of patients

                                Table 1 summarizes the clinicopathological findings of the patients examined. The median follow-up time was 69.0 months (range, 1 to 149 months). The patients included 73 men and 13 women with a median age of 64 years (range, 37 to 81 years). The number of patients in each pathological stage was as follows: 20, pStageI; 28, pStageII; 33, pStageIII; and 5, pStageIV. There were five patients with M1 lymph nodes. Of the 86 patients, 38 (44.2%) were immunohistochemically positive for CD133 (Figure 1). pT and pStage were significantly more advanced among CD133-negative patients compared with CD133-positive patients (Table 1).
                                http://static-content.springer.com/image/art%3A10.1186%2F1477-7819-11-51/MediaObjects/12957_2012_Article_1258_Fig1_HTML.jpg
                                Figure 1

                                Immunohistochemical staining of esophageal squamous cell carcinoma. Tumor cells positive for CD133 (A,B), p16 (C), and p27 (D) expression (×400 magnification). In addition, B, C, and D were at the same site of the same tumor.

                                Table 1

                                Correlation between clinicopathological findings and CD133 status

                                  

                                Total (%)

                                CD133 expression

                                 

                                P

                                   

                                Positive

                                Negative

                                 
                                   

                                n= 38 (44.2%)

                                n= 48 (55.8%)

                                 

                                Age (years)

                                Mean ± SD

                                63.9 ± 9.4

                                64.8 ± 10.9

                                63.2 ± 8.0

                                0.45

                                 

                                (Range)

                                (37–81)

                                (37–9)

                                (44–81)

                                Sex

                                Male

                                73 (84.9)

                                32 (84.2)

                                41 (85.4)

                                0.88

                                Female

                                13 (15.1)

                                6 (15.8)

                                7 (14.9)

                                Smoking

                                Absent

                                13 (15.1)

                                8 (21.1)

                                5 (10.4)

                                0.17

                                Present

                                73 (84.9)

                                30 (79.0)

                                43 (89.6)

                                Location

                                Cervix

                                5 (5.8)

                                3 (7.9)

                                2 (4.2)

                                0.59

                                Upper

                                5 (5.8)

                                1 (2.6)

                                4 (8.3)

                                Middle

                                35 (40.7)

                                17 (44.7)

                                18 (37.5)

                                Lower

                                41 (47.7)

                                17 (44.7)

                                24 (50.0)

                                Histological type

                                Well

                                17 (19.8)

                                9 (23.7)

                                8 (16.7)

                                0.68

                                Moderate

                                56 (65.1)

                                23 (60.5)

                                33 (68.8)

                                Poor

                                13 (15.1)

                                6 (15.8)

                                7 (14.6)

                                pT

                                pT1

                                28 (32.6)

                                19 (50.0)

                                9 (18.8)

                                0.017

                                pT2

                                9 (10.5)

                                3 (7.9)

                                6 (12.5)

                                pT3

                                46 (53.5)

                                15 (39.5)

                                31 (64.6)

                                pT4

                                3 (3.5)

                                1 (2.6)

                                2 (4.2)

                                pN

                                pN0

                                36 (41.9)

                                19 (50.0)

                                17 (35.4)

                                0.17

                                pN1-3

                                50 (58.1)

                                19 (50.0)

                                31 (64.6)

                                pM

                                pM0

                                81 (94.2)

                                35 (92.1)

                                46 (95.8)

                                0.65

                                pM1 (LYM)

                                5 (5.8)

                                3 (7.9)

                                2 (4.2)

                                pStage

                                pStageI

                                20 (23.3)

                                14 (36.8)

                                6 (12.5)

                                0.035

                                pStageII

                                28 (32.6)

                                9 (23.7)

                                19 (39.6)

                                pStageIII

                                33 (38.4)

                                12 (31.6)

                                21 (43.8)

                                pStageIV

                                5 (5.8)

                                3 (7.9)

                                2 (4.2)

                                Lymphatic invasion

                                Negative

                                34 (39.5)

                                13 (34.2)

                                21 (43.8)

                                0.37

                                Positive

                                52 (60.5)

                                25 (65.8)

                                27 (56.3)

                                Venous invasion

                                Negative

                                31 (36.1)

                                11 (29.0)

                                20 (41.7)

                                0.22

                                 

                                Positive

                                55 (64.0)

                                27 (71.1)

                                28 (58.3)

                                 

                                SD, standard deviation.

                                Correlation between CD133 and other markers

                                Table 2 summarizes the correlation between expression of CD133 and expression of other molecular markers examined. CD133 and p27 expression were positively correlated (P = 0.0013), and CD133 and p16 expression tended to be positively correlated (P = 0.057) but did not reach statistical significance. No significant correlations were detected between expression of CD133 and expression of any other marker.
                                Table 2

                                Correlation between expression of CD133 and expression of other molecular markers

                                  

                                Total (%)

                                CD133 expression

                                 

                                P

                                   

                                Positive

                                Negative

                                 
                                   

                                n= 38 (44.2%)

                                n= 48 (55.8%)

                                 

                                p53

                                Negative

                                29 (33.7)

                                13 (34.2)

                                16 (33.3)

                                0.93

                                 

                                Positive

                                57 (66.3)

                                25 (65.8)

                                32 (66.7)

                                p16

                                Negative

                                69 (80.2)

                                27 (71.1)

                                42 (87.5)

                                0.057

                                 

                                Positive

                                17 (19.8)

                                11 (29.0)

                                6 (12.5)

                                p27

                                Negative

                                37 (43.0)

                                9 (23.7)

                                28 (58.3)

                                0.0013

                                 

                                Positive

                                49 (57.0)

                                29 (76.3)

                                20 (41.7)

                                MDM2

                                Negative

                                66 (76.7)

                                31 (81.6)

                                35 (72.9)

                                0.35

                                 

                                Positive

                                20 (23.3)

                                7 (18.4)

                                13 (27.1)

                                Ki-67

                                <30

                                43 (50.0)

                                17 (44.7)

                                26 (54.2)

                                0.39

                                 

                                ≥30

                                43 (50.0)

                                21 (55.3)

                                22 (45.8)

                                EGFR

                                Negative

                                46 (53.5)

                                23 (60.5)

                                23 (47.9)

                                0.24

                                 

                                Positive

                                40 (46.5)

                                15 (39.5)

                                25 (52.1)

                                 

                                Correlations for other molecular markers

                                In terms of correlations between the other molecular markers and clinicopathological findings, p16 expression was correlated with smoking history (P = 0.018), pathological lymph node status (P = 0.033), and lymphatic invasion (P = 0.018) (Additional file1). With regard to correlations among other molecular markers, p53 expression was positively correlated positively with Ki-67 expression (P = 0.0030) (Additional file2).

                                Survival analysis

                                The 3- and 5-year survival rates of all patients examined were 65.0% and 61.5%, respectively. Results of univariate analysis of postoperative overall survival (OS) and disease-free survival (DFS) are summarized in Table 3. Overall survival was significantly correlated with pT, pN, pStage, and CD133 status, and was significantly longer in CD133-positive patients than in CD133-negative patients (P = 0.049) (Figure 2). No significant correlation between OS and the other markers was observed (Figure 3). Multivariate analysis demonstrated that pStage was a significant prognostic factor for OS and that pStage and tumor location were significant prognostic factors for DFS. Correlation between CD133 expression and patient survival did not reach statistical significance by multivariate analysis (Table 4).
                                http://static-content.springer.com/image/art%3A10.1186%2F1477-7819-11-51/MediaObjects/12957_2012_Article_1258_Fig2_HTML.jpg
                                Figure 2

                                Kaplan-Meier curves of patients with esophageal squamous cell carcinoma according to CD133 expression. Overall survival was significantly longer in CD133-positive patients than in CD133-negative patients (P = 0.049). There was no significant correlation between disease-free survival and CD133 status (P = 0.059).

                                http://static-content.springer.com/image/art%3A10.1186%2F1477-7819-11-51/MediaObjects/12957_2012_Article_1258_Fig3_HTML.jpg
                                Figure 3

                                Kaplan-Meier curves of patients with esophageal squamous cell carcinoma according to expression of the other markers. No significant correlation between overall survival and the other markers was observed.

                                Table 3

                                Univariate survival analysis of clinicopathological findings and expression of molecular markers

                                 

                                Variables

                                Number

                                Overall survival

                                Disease-free survival

                                   

                                5-year overall survival rate (%)

                                P

                                5-year disease-free survival rate (%)

                                P

                                Age (years)

                                ≤60

                                29

                                72.4

                                0.11

                                62.1

                                0.21

                                 

                                >60

                                57

                                55.9

                                52.5

                                Sex

                                Male

                                73

                                58.7

                                0.13

                                51.9

                                0.080

                                 

                                Female

                                13

                                76.9

                                76.9

                                Location

                                Cervix/upper 

                                10

                                50.0

                                0.12

                                40.0

                                0.067

                                 

                                Middle/lower 

                                76

                                63.0

                                57.8

                                Histological type

                                Well or moderate

                                73

                                61.5

                                0.64

                                57.4

                                0.36

                                 

                                Poor

                                13

                                61.5

                                46.2

                                pT

                                pT1/pT2

                                37

                                78.1

                                0.0012

                                70.0

                                0.0033

                                 

                                pT3/pT4

                                49

                                49.0

                                44.9

                                pN

                                pN0

                                36

                                80.6

                                0.0051

                                75.0

                                0.0078

                                 

                                pN1-3

                                50

                                47.7

                                41.7

                                pStage

                                pStageI/II

                                48

                                79.0

                                0.0002

                                72.8

                                0.0002

                                 

                                pStageIII/VI

                                38

                                39.5

                                34.2

                                Lymphatic invasion

                                Negative

                                34

                                70.2

                                0.086

                                58.4

                                0.29

                                 

                                Positive

                                52

                                55.8

                                53.9

                                Venous invasion

                                Negative

                                31

                                64.5

                                0.47

                                54.8

                                0.63

                                 

                                Positive

                                55

                                59.8

                                56.2

                                p53

                                Negative

                                29

                                62.1

                                0.30

                                55.2

                                0.55

                                 

                                Positive

                                57

                                61.3

                                56.1

                                p16

                                Negative

                                69

                                58.0

                                0.19

                                53.6

                                0.14

                                 

                                Positive

                                17

                                76.0

                                63.7

                                p27

                                Negative

                                37

                                56.8

                                0.30

                                54.0

                                0.49

                                 

                                Positive

                                49

                                65.0

                                56.9

                                MDM2

                                Negative

                                66

                                63.6

                                0.31

                                59.1

                                0.61

                                 

                                Positive

                                20

                                53.3

                                59.2

                                Ki-67

                                <30

                                43

                                69.8

                                0.24

                                62.8

                                0.27

                                 

                                ≥30

                                43

                                53.2

                                48.6

                                EGFR

                                Negative

                                46

                                69.6

                                0.22

                                63.0

                                0.16

                                 

                                Positive

                                40

                                52.0

                                47.1

                                CD133

                                Negative

                                48

                                54.2

                                0.049

                                47.9

                                0.059

                                 

                                Positive

                                38

                                70.8

                                 

                                65.5

                                 
                                Table 4

                                Multivariate survival analysis of clinicopathological findings and expression of molecular markers

                                 

                                Variables

                                Hazard ratio

                                95% confidence interval

                                P

                                Overall survival

                                pStageIII/IV

                                2.64

                                1.42-5.03

                                0.0020

                                Lymphatic invasion positive

                                1.48

                                0.79-2.89

                                0.23

                                CD133 negative

                                1.74

                                0.94-3.35

                                0.078

                                Disease-free survival

                                Male

                                2.09

                                0.84-6.99

                                0.12

                                 

                                Cervix/upper

                                2.51

                                1.07-5.21

                                0.035

                                 

                                pStageIII/IV

                                2.93

                                1.62-5.42

                                0.0004

                                 

                                CD133 negative

                                1.73

                                0.96-3.24

                                0.071

                                Discussion

                                CD133 was originally identified as a transmembrane glycoprotein in normal hematopoietic stem and progenitor cells[31] that participated in proliferation, self-renewal, and multilineage differentiation[32]. Furthermore, CD133 was recently used to identify putative cancer stem cells of several tumors[33]. According to several studies, CD133 was associated with tumor differentiation in several organs[16, 3436]. For example, Jiang et al.[36] reported that CD133 expression was increased in diffuse-type gastric cancers compared with intestinal-type cancers and was increased more so in poorly differentiated than in moderately or well differentiated gastric cancers. In addition, Feng et al.[35] reported that CD133 was negatively correlated with the cellular differentiation status of colon cancer cells. Finally, Fan et al.[34] reported that CD133 expression was correlated with well differentiated or moderately differentiated cholangiocarcinomas and that subcellular CD133 localization was correlated with the tumor differentiation status. In terms of ESCC, Hang et al. reported that CD133 expression was increased in well differentiated and moderately differentiated ESCCs compared with poorly differentiated ESCCs[30]. However, in our study, no correlation was detected between CD133 expression and tumor differentiation of carcinoma cells. We think that this was because there is a difference among pathologists or facilities regarding the histological evaluation of tumor differentiation status, and in addition, our study was small. On the other hand, CD133 expression correlated with p27 expression (P = 0.0013) and tended to correlate with the status of p16 immunoreactivity (P = 0.057). The relationship between CD133 and cell cycle regulators has remained unclear in esophageal cancer. There may be a correlation between CD133 and cell cycle pathways associated with the INK4 family or the CIP/KIP family of cyclin-dependent kinase inhibitors[37], but this possibility requires further investigation.

                                To the best of our knowledge, there are few reports that have investigated the effect of CD133 expression on survival of ESCC patients. Nakajima et al.[38] reported that CD133 expression in resected ESCC specimens following neoadjuvant chemoradiotherapy tended to be correlated with poor prognosis, but multivariate analysis did not produce a significant correlation. In contrast, CD133 expression was significantly correlated with poor response to neoadjuvant chemoradiotherapy. Hang et al.[30] reported that CD133 expression in resected ESCC specimens without preoperative treatment was not significantly correlated with prognosis. Our study revealed that OS was significantly longer in CD133-positive patients than in CD133-negative patients, as determined by log-rank test. One reason for this was that the tumors were significantly more advanced (according to their pStage classification) in CD133-negative patients than in CD133-positive patients. Although correlation between CD133 expression and patient survival did not reach statistical significance, as determined by multivariate analysis, CD133 immunoreactivity may have the potential to be a good predictor of prognosis in ESCC patients. With regard to other tumors, CD133 expression in non-small cell lung cancers[39, 40], hepatocellular carcinomas[18], and pancreatic cancers[41] was not correlated with patient survival. Moreover, CD133-negative expression in cholangiocarcinomas was correlated with poor prognosis[34], which is similar to that revealed in our study. Further studies are needed to clarify this issue.

                                Conclusions

                                In conclusion, this study demonstrated that CD133 immunoreactivity may have the potential to be a good predictor of prognosis in ESCC patients, and that CD133 may play a role in the regulation of tumor cell cycle through p27 and p16 in ESCC. At present, whether CD133 expression is a valid prognostic marker for ESCC remains controversial. To elucidate this relationship, further investigations are required, including verification of an evaluation method for CD133 immunoreactivity in ESCC.

                                Consent

                                Written informed consent concerning the procedure of this study was obtained from all patients prior to study enrollment.

                                Abbreviations

                                DFS: 

                                Disease-free survival

                                EGFR: 

                                Epidermal growth factor receptor

                                ESCC: 

                                Esophageal squamous cell carcinoma

                                IRS: 

                                Immunoreactive score

                                MDM2: 

                                Murine double minute 2

                                OS: 

                                Overall survival

                                PBS: 

                                Phosphate-buffered saline

                                RT: 

                                Room temperature

                                TNM: 

                                Tumor, node, metastasis

                                UICC: 

                                Union for International Cancer Control.

                                Declarations

                                Acknowledgements

                                We thank the staff of the Department of Pathology, Tohoku University Hospital for their excellent technical assistance and Enago (http://​www.​enago.​jp) for an English language review.

                                Authors’ Affiliations

                                (1)
                                Division of Advanced Surgical Science and Technology, Graduate School of Medicine, Tohoku University
                                (2)
                                Department of Pathology, Tohoku University Hospital
                                (3)
                                Department of Pathology, Graduate School of Medicine, Tohoku University

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