Human Pathology (2014) xx, xxx–xxx

Original contribution

High-mobility group AT-hook 2: an independent marker of poor prognosis in intrahepatic cholangiocarcinoma☆ Chung-Ta Lee MD a , Tsung-Teh Wu MD, PhD b , Christine M. Lohse MS c , Lizhi Zhang MD b,⁎ a

National Cheng Kung University Hospital, Tainan City, Taiwan, 70403, Republic of China Division of Anatomic Pathology, Mayo Clinic, Rochester, MN 55905 c Division of Biomedical Informatics & Statistics, Mayo Clinic, Rochester, MN 55905 b

Received 6 December 2013; revised 25 March 2014; accepted 2 April 2014

Keywords: HMGA2; Intrahepatic cholangiocarcinoma; Prognosis; IHC; Immunohistochemistry; p53

Summary High-mobility group AT-hook 2 (HMGA2) regulates cell growth, differentiation, apoptosis, and neoplastic transformation. Previous studies have shown that malignant tumors expressing HMGA2, such as gastric, lung, and colorectal carcinomas, usually have a poor prognosis. HMGA2 expression and its clinical significance in intrahepatic cholangiocarcinomas have not been studied. We identified 55 intrahepatic cholangiocarcinomas resected at our institution from 1994 to 2003. Hematoxylin-eosin–stained slides were reviewed, and histopathologic characteristics were recorded, including mitotic count, tumor grade, vascular and perineural invasion, lymph node metastasis, and margin status. Using immunohistochemical stains, we examined expression of HMGA2, p53, p16, Kit, α-fetoprotein, and Ki-67, and we analyzed the correlation of survival with clinicopathological characteristics and immunohistochemical findings. Positive staining for HMGA2, p53, p16, Kit, α-fetoprotein, and Ki-67 was seen in 18 (33%), 37 (69%), 26 (47%), 21 (38%), 2 (4%), and 34 (63%) tumors, respectively. HMGA2 expression correlated positively with p53 expression (P = .02; ρ = 0.32) and negatively with p16 expression (P = .04; ρ = −0.28). Univariate analysis showed that HMGA2 expression and lymph node metastasis were associated with shorter patient survival and were independent indicators of poor survival (P = .02 and P = .03, respectively). Tumorigenic effects of HMGA2 in intrahepatic cholangiocarcinoma may partly reflect its ability to negatively regulate expression of p16 tumor suppressors and to be associated with p53 abnormalities. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Intrahepatic cholangiocarcinoma arising in the intrahepatic biliary tree is the second most common primary hepatic Abbreviations AFP, α-fetoprotein; HMGA2, high-mobility

group AT-hook 2. ☆

Competing interest: None. ⁎ Corresponding author. Division of Anatomic Pathology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail address: [email protected] (L. Zhang). 0046-8177/© 2014 Elsevier Inc. All rights reserved.

malignancy [1]. In Western countries, most cases occur after age 60 years and without significant associated cirrhosis. Clinically, abdominal pain and weight loss are the predominant symptom and sign [2]. However, intrahepatic cholangiocarcinomas without obstruction of the central bile duct usually remain asymptomatic until they attain a large size, which renders surgical removal difficult or impossible. Intrahepatic cholangiocarcinoma is associated with a high mortality rate because of early invasion, widespread metastasis, and lack of effective nonsurgical therapeutic methods [3]. The

2 overall survival rate, including that in patients who undergo resection, is poor, with less than 5% of patients surviving 5 years [4,5]. The negative prognostic factors include macroscopic periductal infiltration, macroscopic vascular invasion, positive surgical margins, advanced TNM stage, lymph node spread, and intrahepatic metastasis [6,7]. In addition, certain molecular biomarkers have been reported to correlate with poor survival and tumor progression, such as MUC1, MUC4, fascin, and epidermal growth factor receptor [8–11], but most of them are not used routinely in clinical practice. HMGA2, previously known as high-mobility group protein isoform I-C, belongs to the nonhistone chromosomal high-mobility protein family. The HMGA2 protein is an architectural nuclear factor containing structural DNA-binding domains and may act as a transcription-regulating factor. It is highly expressed during embryogenesis but is scanty or absent in normal adult tissues [12], in which it may function as an oncogene. Overexpression of HMGA2 has been identified in various human cancers [13–26], including gastric, pancreatic, colorectal, lung, and ovarian cancers, and its overexpression is correlated with high tumor grade [16,19,21,23,25], lymph node metastasis [16,21,23], and poor prognosis [13,15,20,23]. Our previous study showed that HMGA2 also is overexpressed in hepatoblastoma [27]. The HMGA2 protein could exert its oncogenicity in several ways, such as activation of the cell cycle and modulation of apoptosis [28]. The protein governs proliferation and differentiation and seems to be related to the self-renewing activity of stem cells by decreasing p16 expression and cellular senescence [29,30], a finding that might shed light on oncogenesis. The p16 protein is a cell-cycle inhibitor and a well-known tumor suppressor gene. It promotes cellular senescence; lack of p16 therefore allows for inappropriate progression to S phase [31]. The expression of p16 usually is low in intrahepatic cholangiocarcinomas associated with hepatolithiasis [32]. In a multivariate analysis, Karamitopoulou et al [33] showed that loss of p16 expression was associated with reduced survival of cholangiocarcinoma patients. This finding has not yet been confirmed by other studies. The p53 protein also is a classic cell-cycle regulator at the G1-S checkpoint and a highly important tumor suppressor gene in humans. It is involved in controlling DNA repair and apoptosis [34]. Inactivation of p53 occurs in most human cancers by one of several mechanisms, which all generally lead to nuclear accumulation of the protein [35]. Mutations of TP53 are present in 28% to 61% of cholangiocarcinomas [36] and were implicated in the genesis of cholangiocarcinoma in various immunohistochemical and molecular epidemiologic investigations. Abnormal expression of p53 is seen in 19% to 86% of cholangiocarcinomas on immunostaining [35]. Some studies, including one showing independent prognostic significance, found an association between p53 overexpression and decreased overall survival; however, another study demonstrated no association with outcome [36]. Expression of HMGA2 in intrahepatic cholangiocarcinomas and its clinical significance have not been studied. In the present

C. -T. Lee et al. study, we examined HMGA2 expression, as well as various clinicohistologic characteristics, including the expression of p16, p53, and 2 embryogenic proteins, Kit and AFP, in intrahepatic cholangiocarcinomas and correlated the findings with patient survival.

2. Materials and methods 2.1. Patients and materials This study was approved by the Mayo Clinic Institutional Review Board. We identified 68 patients with intrahepatic cholangiocarcinoma through a search of the Mayo Clinic's electronic pathology database of patients seen between 1995 and 2003. All intrahepatic cholangiocarcinomas had been resected. Thirteen patients were lost to follow-up and thus were excluded from this study. The histologic characteristics of all cases were reviewed and the diagnoses confirmed. The clinicopathological characteristics of these patients are summarized in Table 1.

2.2. Histologic evaluation The histologic characteristics evaluated included histologic tumor grade, lymph node metastases, surgical margin status, perineural invasion, vascular invasion, necrosis, signet ring tumor cells, bile duct dysplasia, mitoses, desmoplasia, and status of the tumor–normal tissue interface. The gross features of the tumors such as macroscopic periductal infiltration or macroscopic vascular invasion were not considered because of inconsistency in the older pathology reports. The histologic grade of intrahepatic cholangiocarcinomas was classified on the basis of the proportion of tumor composed of glands [37]: • • • •

Grade Grade Grade Grade

1, 2, 3, 4,

N95% 50%-95% 5%-49% b5%

Surgical margins involved by tumor cells were counted as positive; those not involved by tumor cells were counted as negative. Bile duct dysplasia was examined in available slides from nonadjacent liver or hilar tissue to avoid inappropriate diagnosis of cancer of the bile duct. When the characteristics of neural invasion, vascular invasion, necrosis, signet ring tumor cells, or bile duct dysplasia were present, the case was counted as positive for these features; if these features were not present, the case was counted as negative. The tumor–normal tissue interface was defined as expansile or infiltrative. Intrahepatic cholangiocarcinomas with an expansile interface formed a well-demarcated nodule with growth in a radial pattern and invasion of the adjacent liver parenchyma. By comparison, the infiltrative cholangiocarcinoma grows in a diffuse longitudinal pattern along

HMGA2 in cholangiocarcinoma


Table 1 Clinicopathological characteristics and univariate associations with death in intrahepatic cholangiocarcinomas Characteristic a

Value b

Age at operation (y), 60.9 (12.3) mean (SD; n = 55) Sex (n = 55) Female 34 (62) Male 21 (38) Tumor size (cm), 7.9 (3.6) mean (SD; n = 52) Desmoplasia, mean (SD) 42.0 (16.1) Mitoses per 10 HPF, 12.2 (9.3) mean (SD) Satellite 0 36 (68) N0 17 (32) Tumor grade 2 8 (15) 3 38 (72) 4 7 (13) Tumor stage 1 14 (26) 2 21 (40) 3 7 (13) 4 11 (21) Signet ring cell features 8 (15) Perineural invasion 8 (15) Vascular invasion 9 (17) Necrosis 30 (57) Lymph node metastases 12 (23) Bile duct dysplasia 12 (23) Positive margins 4 (8) Interface Expansion 13 (25) Infiltration 40 (75)

Death, HR (95% CI)


1.04 (0.78-1.39) c


1.0 (reference) 0.88 (0.46-1.67) .69 1.05 (0.96-1.15) d .32 0.94 (0.78-1.14) e 1.31 (0.91-1.89) e

.55 .15

1.0 (reference) 1.78 (0.94-3.37)


1.0 (reference) 1.16 (0.48-2.80) 0.63 (0.18-2.25)

.74 .48

(diluted 1:2000; Dako, Denmark), and anti-Ki-67 (diluted 1:20; Dako, Denmark). Briefly, 4-μm-thick sections were cut from paraffin-embedded tissue blocks. After deparaffinization and rehydration, antigen retrieval was performed. After the avidin-biotin complex procedure, immunoreactions were developed using 0.03% 3,3′-diaminobenzidine tetrahydrochloride, and sections were counterstained with Harris hematoxylin. Positive and negative control samples were included in all runs. The immunohistochemistry assessments were made by 2 pathologists (C. T. L. and L. Z.). Positivity of the proteins was semiquantified as percentage of positive cells among total tumor cells. For HMGA2, p16, p53, and Ki-67, nuclear positivity was counted; for Kit and AFP, cytoplasmic positivity was counted. The immunohistochemistry results of HMGA2, p16, p53, Kit, and AFP were grouped into less than 5% (negative), 5% to 49% (focally positive), and 50% and greater (diffusely positive) [24,33,39]. Results of Ki-67 were grouped into less than 1% (negative) and 1% or greater (positive).

2.4. Statistical analysis 1.0 (reference) 1.47 (0.66-3.31) 2.09 (0.69-6.38) 1.29 (0.49-3.38) 0.73 (0.29-1.87) 0.70 (0.29-1.68) 0.66 (0.29-1.51) 0.84 (0.45-1.59) 2.38 (1.14-4.96) 0.73 (0.34-1.59) 2.34 (0.81-6.82)

.35 .19 .60 .51 .43 .33 .59 .02 .43 .12

1.0 (reference) 0.84 (0.43-1.66)


Abbreviations: HR, hazard ratio; CI, confidence interval; HPF, highpower fields. a Values are expressed for 53 patients unless specified otherwise. b Values are presented as number and percentage of patients unless specified otherwise. c Ten-year increase. d One-centimeter increase. e Ten-unit increase.

Continuous characteristics were summarized with mean (SD); categorical characteristics were summarized with frequency count and percentage. The duration of follow-up was calculated from the date of surgery to the date of death or last follow-up. Associations with overall survival were evaluated through Cox proportional hazards regression models and summarized using the hazard ratio (HR) and 95% confidence interval (CI). A multivariate model was built through stepwise selection, with the P set at .05 for a characteristic to enter or leave the model. Statistical analyses were performed with the SAS software package Version 9.2 (SAS Institute Inc, Cary, NC). The survival curve was calculated using the Kaplan-Meier method. The relation between the immunohistochemistry reactivity of HMGA2 and other proteins was analyzed and quantified using the Spearman rank sum correlation coefficient ρ. All tests were 2 sided, and P b .05 was considered statistically significant.

3. Results the bile duct [37,38]. We counted the number of mitoses per 10 high-power fields and calculated the percentage of desmoplasia in the whole tumorous tissue on the slide.

2.3. Immunohistochemistry staining Immunohistochemistry staining was performed using a standard avidin–biotin complex-peroxidase procedure by an automated stainer (Dako, Denmark). The primary antibodies were anti-HMGA2 (diluted 1:1000; Biocheck, Foastr City, CA), anti-p16 (diluted 1:1000; Ventana Medical Systems, Tucson, AZ), Kit (diluted 1:500; Dako), anti-AFP (diluted 1:150; Ventana Medical Systems, Tucson, AZ), anti-p53

3.1. Clinicopathological and immunohistochemical staining characteristics The clinicopathological characteristics of the 21 men and 34 women with intrahepatic cholangiocarcinoma are summarized in Table 1. The mean age of the patients at surgery was 60.9 years. The mean tumor size was 7.9 cm. Seventeen (32%) of 53 patients had 1 or more satellite tumor nodules. Eight (15%), 38 (72%), and 7 (13%) intrahepatic cholangiocarcinomas were tumor grades 2, 3, and 4, respectively. Fourteen (26%), 21 (40%), 7 (13%), and 11 (21%) intrahepatic cholangiocarcinomas were stages 1, 2, 3, and 4, respectively.


C. -T. Lee et al.





Fig. 1 HMGA2, p53, and p16 expression in intrahepatic cholangiocarcinoma. A, Hematoxylin–eosin stain of intrahepatic cholangiocarcinoma. B, Tumor cells positive for HMGA2. C, Tumor cells positive for p53. D, Tumor cells negative for p16. All images, original magnification ×200.

Twelve (23%) of the 53 intrahepatic cholangiocarcinomas were associated with lymph node metastases. Four (8%) intrahepatic cholangiocarcinomas had tumor invading the surgical margins. A hematoxylin-eosin stain of a representative case is shown in Fig. 1A. Results of immunohistochemistry staining are summarized in Table 2. HMGA2, p16, p53, and Ki-67 proteins were expressed in the nuclei of tumor cells; Kit proteins were expressed in the cytoplasm, cell membrane, or both; AFP was expressed in the cytoplasm. According to our criteria, positive staining for HMGA2 (Fig. 1B), p53 (Fig. 1C), p16, Kit, and Ki-67 was seen in 18 (33%), 37 (69%), 26 (47%), 21 (38%), and 34 (63%) of tumors, respectively. Only 2 tumors (4%) were positive for AFP.

3.2. Correlation between HMGA2 expression and other clinicopathological characteristics Expression of HMGA2 correlated positively with p53 expression (P = .02; ρ = 0.32) and correlated negatively with p16 expression (P = .04; ρ = −0.28). There was a nonsignificant tendency of HMGA2 expression to correlate positively with Ki-67 expression (P = .09; ρ = 0.24). In Fig. 1, we show an area of intrahepatic cholangiocarcinoma that is positive for HMGA2 and p53 and negative for p16. Statistical significance was found between HMGA2 expression and the tumor–normal tissue interface (P = .03); of HMGA2-positive intrahepatic cholangiocarcinomas, 94% had an infiltrating tumor–normal tissue interface. The correlation between

HMGA2 expression and the other clinicopathological features is summarized in Supplementary Figure and Tables 1 and 2.

3.3. Survival and its correlation with various clinicopathological characteristics At last follow-up, 42 patients had died (mean survival after surgical procedure, 3.5 years; median [range], 2.8 years [12 days–12.2 years]). The mean duration of follow-up for the 13 patients who were still alive at last follow-up was 7.7 years (8.0 years [50 days–15.2 years]). Univariate associations of the clinicopathological characteristics with mortality are summarized in Tables 1 and 2. The association of AFP with mortality is not given because only 2 tumors expressed this protein. The immunohistochemistry characteristics of HMGA2, p53, p16, and Kit were analyzed as less than 5% (negative) versus 5% or greater (positive). Among these clinicopathological characteristics, only lymph node metastases and the expression of HMGA2 influenced patient survival significantly. Patients with lymph node metastases had about 2.38 times the risk of death (P = .02) as patients without lymph node metastasis; patients with HMGA2-positive intrahepatic cholangiocarcinomas had about 2.20 times the risk of death (P = .02) as patients with HMGA2-negative tumors. The univariate association of HMGA2 with overall survival by the Kaplan-Meier method is illustrated in Fig. 2 (P = .01). Table 3 summarizes the multivariate model. Jointly, HMGA2 and lymph node metastases were significantly

HMGA2 in cholangiocarcinoma


Table 2 Expression of biomarkers in 55 intrahepatic cholangiocarcinomas Characteristic HMGA2 Negative Positive Focal Diffuse p53 (n = 54) Negative Positive Focal Diffuse p16 Negative Positive Focal Diffuse Kit Negative Positive Focal Diffuse AFP (n = 54) Negative Positive Focal Diffuse Ki-67 (n = 54) Negative Positive

Table 3

n (%)

Death, HR (95% CI)

37 (67)

1.0 (reference) 2.20 (1.15-4.22)



9 (16) 9 (16) 17 (31)

1.0 (reference) 1.0 (0.51-1.96)


HMGA2 positive Lymph node metastases

2.20 (1.12-4.33) 2.28 (1.08-4.81)

.02 .03

Abbreviations: HR, hazard ratio; CI, confidence interval.

4. Discussion

29 (53)

1.0 (reference) 0.82 (0.45-1.51)


1.0 (reference) 0.69 (0.36-1.31)


1.0 (reference) 1.50 (0.77-2.89)


20 (36) 6 (11) 34 (62) 17 (31) 4 (7) 52 (96) 2 (4) 0 20 (37) 34 (63)

associated with overall survival. Patients with HMGA2positive intrahepatic cholangiocarcinomas had 2.20 times the risk of death as patients with HMGA2-negative tumors (P = .02). After adjustment for this characteristic, the patients 100


Overall Survival

HR (95% CI)


22 (41) 15 (28)

HMGA2 negative (n = 37)



20 HMGA2 positive (n = 18) P = 01

0 1


with lymph node metastases had 2.28 times the risk of death as patients without lymph node metastases (P = .03).

Abbreviations: HR, hazard ratio; CI, confidence interval. a Immunohistochemical staining results were categorized into positive and negative for each comparison.


Multivariate associations with mortality in 53 patients





Years From Surgery

Fig. 2 Kaplan-Meier estimates of overall survival in relation to HMGA2 expression in intrahepatic cholangiocarcinoma (P = .01).

In our study, HMGA2 expression was observed in 18 (33%) of intrahepatic cholangiocarcinomas and was associated with shorter patient survival. The HMGA2 protein is an architectural nuclear factor involved in chromatin remodeling and gene transcription. It is highly expressed during embryogenesis, but its expression is low or absent in normal adult tissues [12]. Recently, HMGA2 has been found to be overexpressed in various human cancers [13–26] and to be more frequently seen in cancers with a more aggressive phenotype, such as ovarian or endometrial high-grade serous carcinoma [17,22,25]. Expression correlated with increasing tumor grade [16,19,21,23,25], advanced stage [19,22], vascular invasion [24], serosal invasion [15], lymph node metastasis [16,21,23], and distant metastasis [20]. The protein is an independent indicator of tumor progression in bladder cancer [19] and of shorter patient survival in gastric cancer and oral squamous cell carcinoma [13,15]. These data and our results support a role for HMGA2 in the progression of many cancer types. The protein thus could be a useful biomarker and a rational therapeutic target in intrahepatic cholangiocarcinoma. The HMGA2 protein could exert its oncogenicity in several ways, such as activation of the cell cycle, enhancement of transcriptional factor activity, interference with apoptosis, and impairment of DNA repair [28]. The role of high-mobility group A proteins in cell transformation is based on their ability to down-regulate or up-regulate expression of genes that have a critical role in the control of cell proliferation and invasion [28]. The HMGA2 protein seems to be related to the selfrenewing activity of stem cells by decreasing p16 expression and cellular senescence [29,30]. Yu et al [29] showed that HMGA2 enhanced proliferation by inducing pI3K/AKT/ mTOR/p70S6K signaling and inhibiting p16INK4A and p21CIP1/WAF1 expression in human umbilical cord blood– derived stromal cells. Nishino et al [30] showed that HMGA2 contributed to neural stem cell properties by repressing the expression of p16INK4A and p19ARF . In agreement with the literature, we found that HMGA2 protein expression was negatively correlated with p16 protein expression in intrahepatic cholangiocarcinoma—supportive of the possibility that HMGA2's tumorigenic effects may reflect in part its ability to regulate negatively the expression of the p16 tumor suppressors.

6 In our experiments, HMGA2 expression was positively correlated with p53 expression in intrahepatic cholangiocarcinoma. TP53 is a highly important tumor suppressor gene [34], and its inactivation as a result of mutation generally leads to nuclear p53 protein accumulation [35]. Although the mechanism is unclear, the positive correlation between HMGA2 and p53 expression in our study is in agreement with the results in high-grade serous carcinomas of the ovaries and fallopian tubes [17,18]. The discovery suggests that HMGA2 overexpression is an important biomarker in conjunction with p53 for the diagnosis of intrahepatic cholangiocarcinomas and possibly is associated with tumorigenesis. Further study characterizing the relation between TP53 mutations and HMGA2 expression will be valuable for understanding the functional role of HMGA2 in tumorigenesis of intrahepatic cholangiocarcinoma. The HMGA2 protein is highly expressed during embryogenesis but is low or absent in normal adult tissues [12]. Therefore, we also tested 2 other embryogenic proteins found in the liver, namely, AFP and Kit, but their expression was not associated with patient prognosis. We evaluated various histologic features and investigated their correlation with the survival of patients who had intrahepatic cholangiocarcinoma. Consistent with the literature, patients with lymph node metastases had a shorter survival than did those without such metastases. As expected, multiple tumors, macroscopic periductal infiltration, vascular invasion, and positive surgical margins are also important negative prognostic factors in intrahepatic cholangiocarcinoma [6,7], but a correlation with patient survival was lacking in our study, which may be attributable to the small number of cases. The clinical significance of signet ring tumor cells in intrahepatic cholangiocarcinoma had not been studied before, and our results showed that they did not affect patient survival. In conclusion, we found that HMGA2 protein expression was correlated with a shorter overall survival in patients with intrahepatic cholangiocarcinomas. Thus, HMGA2 could be a useful biomarker and therapeutic target in this disease. The tumorigenic effects of HMGA2 in intrahepatic cholangiocarcinoma may partly reflect its ability to regulate the expression of p16 tumor suppressors negatively and to be associated with p53 abnormalities. Further studies are needed to elucidate this possibility.

Supplementary data Supplementary data to this article can be found online at

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Supplementary figures. Supplementary tables.

High-mobility group AT-hook 2: an independent marker of poor prognosis in intrahepatic cholangiocarcinoma.

High-mobility group AT-hook 2 (HMGA2) regulates cell growth, differentiation, apoptosis, and neoplastic transformation. Previous studies have shown th...
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