Abstract
Objectives:
Cervical cancer is one of the most common gynecological malignancies worldwide, and its association with the AMP-activated protein kinase (AMPK) is still unknown. We aimed to investigate the clinical correlation between AMPK expression and cervical cancer.
Methods:
The expression of AMPKα1, AMPKα2 and phosphorylated AMPKα (p-AMPKα) was determined immunohistochemically in 524 formalin-fixed, paraffin-embedded malignant and premalignant cervical tissues. Subsequently, associations with clinicopathological characteristics and patient survival were assessed.
Results:
AMPKα2 expression was observed in the cytoplasm and nucleus, while expression of AMPKα1 and p-AMPKα was mainly observed in the cytoplasm. p-AMPKα expression increased during the normal-to-tumor transition of cervical carcinoma (p < 0.001), but, once cancer developed, the expression of AMPKα2 and p-AMPKα decreased in large-sized tumors when compared to smaller tumors (36 vs. 68%, p = 0.004 and 39 vs. 64%, p = 0.029, respectively). Notably, AMPKα2 expression was significantly associated with better disease-free survival (HR 0.29, 95% CI 0.10–0.86, p = 0.026).
Conclusion:
The AMPKα2 isoform showed potential as a favorable prognostic marker in cervical cancer. Therefore, additional studies are necessary to further clarify the complex contribution of AMPK isoforms and of phosphorylation status to cervical cancer progression and prognosis.
Keywords: AMP-activated protein kinase, Carcinogenesis, Cervical neoplasms, Disease-free survival
Introduction
Cervical cancer is one of the most common gynecological cancers worldwide. Although persistent human papillomavirus (HPV) infection plays a significant role in the initiation of cervical neoplasia, it is not sufficient for cervical carcinogenesis [1]. Other factors, such as the upregulation of oncogenes and related signaling pathways, may be involved in cervical carcinogenesis [2]. Once cancer develops, several clinical factors such as International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis and tumor size can be used as markers for prognosis; they are not, however, sufficient. Therefore, new markers including biological predictors are needed for accurate prognosis in individual patients.
AMP-activated protein kinase (AMPK) is the major regulator of cellular metabolism and energy homeostasis [3–8]. It is activated typically by an increase in the cellular AMP/adenosine triphosphate (ATP) ratio under conditions such as hypoxia, nutrient deprivation and heat shock [9]. Prior studies have shown that pharmacological activators of AMPK exert antitumor effects in numerous cancers [3, 6, 10, 11]. The liver kinase B1 (LKB1)/AMPK/mTOR signaling pathway is thought to mediate the tumor suppressive effect of pharmacological agents such as metformin and AICAR [12–14]. Indeed, LKB1 can mediate the action of metformin on AMPK activity [3]. Recently, Xiao et al. [15] showed that metformin could induce both apoptosis and autophagy in cervical cancer cells when LKB1 is expressed. Notably, frequent mutation of LKB1 has been identified in cervical cancer [5], and one of the best-characterized substrates of LKB1 is AMPK [16]. In addition, several clinical studies have shown that expression of AMPK is associated with tumor grade and prognosis for various solid tumors, e.g. those occurring in the colorectum, breast, ovary and prostate [17–19]. However, knowledge about the role of AMPK in tumor progression and its clinical significance in cervical cancer is limited.
AMPK is a heterotrimeric protein composed of a catalytic α subunit and 2 regulatory subunits, β and γ. The α subunit, containing a serine/threonine protein kinase catalytic domain, has 2 isoforms, i.e. AMPKα1 and AMPKα2 [20]. Although they are highly homologous, it is becoming increasingly clear that AMPKα1 and AMPKα2 are encoded by 2 distinct genes that share mutual and exclusive functions, and they have different tissue distribution and cellular localization [21–23]. Nonetheless, little is known about the isoforms and phosphorylation status of AMPKα and its significance in cervical carcinoma. In this study, we investigated the expression of AMPKα isoforms and their prognostic value in cervical cancer patients.
Materials and Methods
Patients and Specimens
One hundred and twenty-four cervical cancer tissues, 302 high-grade cervical intraepithelial neoplasia (CIN) samples, 98 low-grade CIN samples and 100 normal cervical epithelia were prospectively collected from patients who were admitted to the Gangnam Severance Hospital, Yonsei University College of Medicine (Seoul, South Korea) and underwent primary surgery in the period from 1996 to 2010. Some of the paraffin blocks were provided by the Korea Gynecologic Cancer Bank through the Bio and Medical Technology Development Program of the Ministry of Education, Science and Technology, Korea (NRF-2012M3A9B8021800). Clinical and follow-up data were obtained from patients’ medical records. Cervical cancer was staged according to the FIGO staging system and graded according to the World Health Organization (WHO) grading system. Adjuvant radiotherapy with or without concurrent chemotherapy was administered if the patient had risk factors for recurrence. Disease-free survival (DFS) was assessed from the date of surgery to the date of recurrence or the date of the last follow-up visit. Tissue samples were collected from patients who provided informed consent. This study was approved by the Institutional Review Board of Gangnam Severance Hospital and additionally approved by the Office of Human Subjects Research at the National Institutes of Health.
Western Blotting
Two cervical cancer cell lines, HeLa and CaSki, were cultured, harvested and then fractionated with the NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer’s protocol (NE-PER Reagents, Thermo Scientific, Rockford, Ill., USA). Twenty micrograms of the cellular fractions were separated by 4–12% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking for 1 h with 5% nonfat milk in TBST (50 mM Tris, 150 mM NaCl and 0.05% Tween 20; pH 7.5), the membrane was probed with the following primary antibodies: rabbit polyclonal anti-AMPKα1 antibodies (Cell Signaling, Danvers, Mass., USA; Cat. No. 2795, 1: 1,000 dilution), rabbit polyclonal anti-AMPKα2 antibodies (Cell Signaling; Cat. No. 2757, 1: 1,000 dilution) and rabbit monoclonal anti-phosphorylated (p)-AMPKα antibodies (Cell Signaling; Cat. No. 2535, 1: 500 dilution). The membrane was incubated with the appropriate secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized by a SuperSignal chemiluminescence kit (Thermo Scientific). Calnexin (BD Transduction Lab, San Jose, Calif., USA; Cat No. 610523, 1: 1,000 dilution) and lamin B1 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA; Cat No. sc-20682, 1: 1,000 dilution) were used as cytoplasm and nuclear extract controls, respectively.
Tissue Microarray and Immunohistochemistry
Tissue microarrays were constructed from 624 formalin-fixed, paraffin-embedded tissue blocks after being reviewed by an experienced pathologist (S.M.H.). Three 1.0-mm-diameter tissue cores were arrayed on a recipient paraffin block using a tissue arrayer (Pathology Devices, Westminster, Md., USA). Areas where tumor cells occupied >60% of cells with major histological differentiation were selected.
Immunohistochemical staining was performed on 5-micronthick sections. The tissue sections were deparaffinized with xylene and dehydrated with series of graded ethanol. Heat-induced antigen retrieval was conducted for 20 min in antigen-retrieval buffer (pH 9.0; Dako, Carpinteria, Calif., USA) using a steam pressure cooker (Pascal, Dako). The specimens were incubated with 3% H2O2 for 10 min to block endogenous peroxidase. Nonspecific binding was blocked with protein block (Dako) for 20 min at room temperature. The sections were incubated at 4 °C overnight with rabbit polyclonal anti-AMPKα1 antibodies (Cell Signaling; Cat. No. 2795) at 1: 400 dilution, rabbit polyclonal anti-AMPKα2 antibodies (Cell Signaling; Cat. No. 2757) at 1: 200 dilution and rabbit monoclonal anti-p-AMPKα antibodies (Cell Signaling; Clone No. 40H9, Cat. No. 2535) at 1: 200 dilution, respectively. The antigen-antibody reaction was detected with EnVision+ Dual Link System-HRP (Dako) and visualized with DAB+ (3,3′-diaminobenzidine; Dako). Tissue sections were lightly counterstained with hematoxylin and then examined by light microscopy. Negative controls (substitution of primary antibody with TBS) were run simultaneously. Positive controls included normal spleen, breast and gastric epithelium for the AMPKα1/AMPKα2/p-AMPKα antibodies, respectively.
Digital Image Analysis
The stained sections were digitized utilizing the NanoZoomer 2.0 HT (Hamamatsu Photonics K.K., Japan) at ×20 objective magnification (0.5 μm resolution). The images were analyzed using Visiopharm software v4.5.1.324 (Visiopharm, Horsholm, Denmark). The threshold for size and shape of tumor cells was manually calibrated. Briefly, brown-colored (DAB) and blue-colored (hematoxylin) cells were separated spectrally. A brown staining intensity (0 = negative, 1 = weak, 2 = moderate and 3 = strong) was obtained using a predefined algorithm and optimized settings. The overall immunohistochemical score (histoscore) was expressed as the percentage of positive cells multiplied by their staining intensity (possible range 0–300). The cut-off values of histoscores were defined by considering the distribution and prognostic significance of the values (online suppl. fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000434726).
Statistical Analysis
The Mann-Whitney U test and χ2 test were used to compare the AMPK expression in the different groups. Survival distributions were estimated using the Kaplan-Meier method, and the relationship between survival and each parameter was analyzed with the log-rank test. Multivariate analyses with hazard ratio (HR) for recurrence were performed using the Cox proportional-hazards regression. Statistical analyses were performed using SPSS v21.0 (SPSS Inc., Chicago, Ill., USA). A value of p < 0.05 was considered statistically significant.
Results
Clinicopathological Characteristics of Patients
Clinicopathological characteristics of the patients were summarized in table 1. The mean age of the patients was 42.1 ± 12.0 years. Of the 124 patients with cervical cancer, 111 (89.5%) had stage I and 13 (10.5%) had stage II. One hundred and one (83.5%) patients had squamous-cell carcinoma and in 81 (69.2%), these were well/moderately differentiated. Twenty-eight patients (22.6%) with a tumor size ≥ 4 cm were categorized as the large-sized tumor group. Lymph node metastasis was found in 19 (15.6%) patients.
Table 1.
Clinicopathological characteristics
| Characteristic | Frequency | % |
|---|---|---|
| Diagnostic category | ||
| Normal | 100 | 16.0 |
| Low-grade CIN | 98 | 15.7 |
| High-grade CIN | 302 | 48.4 |
| Cervical cancer | 124 | 19.9 |
| FIGO stage | ||
| I | 111 | 89.5 |
| II | 13 | 10.5 |
| Differentiation | ||
| Well/moderate | 81 | 69.2 |
| Poor | 36 | 30.8 |
| Cell type | ||
| Squamous-cell carcinoma | 101 | 83.5 |
| Adenocarcinoma | 20 | 16.5 |
| Tumor size | ||
| <4 cm | 96 | 77.4 |
| ≥4 cm | 28 | 22.6 |
| Lymphovascular invasion | ||
| Negative | 64 | 64.0 |
| Positive | 36 | 36.0 |
| Lymph node metastasis | ||
| Negative | 103 | 84.4 |
| Positive | 19 | 15.6 |
Expressions and Cellular Localizations of AMPK
To examine the specificity and affinity of anti-AMPKα1, anti-AMPKα2 and anti-p-AMPKα antibodies, we investigated AMPKα1, AMPKα2 and p-AMPKα expression using fractionated CaSki and HeLa cell lysates. AMPKα1 and p-AMPKα were predominantly detected in the cytosolic fraction, and AMPKα2 was detected in the cytosolic and nuclear fractions (fig. 1). The purities of the cytosolic and nuclear fractions were confirmed with calnexin and lamin B1, respectively.
Fig. 1.
Subcellular localization of AMPKα1, AMPKα2 and p-AMPKα in cervical cancer cell lines. Cytosolic and nuclear fractions from CaSki and HeLa cells were analyzed by Western blot analysis. Calnexin and lamin B1 were used as an index for the cytosolic and nuclear fractions, respectively.
The tissue microarrays contained 124 cases of cervical cancer; however, due to the complexity of sectioning and staining as well as the heterogeneity of the samples, only 111, 115 and 122 samples could be interpreted for AMPKα1, AMPKα2 and p-AMPKα, respectively. Representative staining of AMPKα1, AMPKα2 and p-AMPKα is shown in figure 2. In general, AMPKα2 expression was observed in the cytoplasm and nucleus, and AMPKα1 and p-AMPKα expression was mainly observed in the cytoplasm (fig. 2).
Fig. 2.
Representative immunohistochemical staining of AMPKα1, AMPKα2 and p-AMPKα expression in human cervical neoplasms. a Negative for AMPKα1 expression. b Positive for AMPKα1 cytoplasmic expression. c Negative for AMPKα2 expression. d Positive for AMPKα2 cytoplasmic and nuclear expression. e Negative for p-AMPKα expression. f Positive for p-AMPKα cytoplasmic expression. Scale bar: 100 μm.
Levels of AMPKs expression during cervical carcinogenesis are depicted in figure 3. p-AMPKα expression showed a gradual increase during tumorigenesis. The expression of p-AMPKα in cancer and high-grade CIN was higher than that in low-grade CIN and normal cervical tissue (p < 0.001, respectively). However, differential expression of AMPKα1 was only found between cancer tissues and high-grade CIN (median histoscore 110 vs. 100; p < 0.01). The expression of AMPKα2 was not different during tumor progression.
Fig. 3.
Expression of AMPKα1, AMPKα2 and p-AMPKα1/2 during cervical carcinogenesis. a Histoscore for AMPKα1 was significantly higher in cervical cancer specimens than in high-grade CIN. The histoscore for AMPKα2 was not significantly different (b) but that for p-AMPKα was higher in CIN and tumor specimens than in normal tissues (c). * p < 0.05, * * p < 0.01. HG = High-grade; LG = low-grade.
To examine the prognostic value of AMPK isoforms as biomarkers in the transition from high-grade CIN to cancer, we further defined the subgroups for analysis (online suppl. fig. 2). The expression of AMPKα1 and p-AMPKα in the HPV-positive group was significantly higher in cancer (p = 0.035 and p = 0.010, respectively) than in high-grade CIN whereas in the HPV-negative group, there was no statistical difference between high-grade CIN and cancer.
The association between AMPK expression and clinicopathological characteristics in cervical cancer patients is summarized in table 2. Sixty-eight out of 111 patients (61.3%) were categorized as AMPKα1-positive, 69/115 (60.0%) as AMPKα2-positive and 71/122 (58.2%) as p-AMPKα-positive. AMPKα2 and p-AMPKα expression was less frequent in large-sized tumor tissues than in small-sized tumor tissues (36 vs. 68%, p = 0.004 and 39 vs. 64%, p = 0.029, respectively). There were no other correlations between AMPK expression and clinicopathological characteristics.
Table 2.
Correlation between AMPK expression and clinicopathological characteristics of cervical cancer
| AMPKα1 |
AMPKα2 |
p-AMPKα |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| negative | positive | p value | negative | positive | p value | negative | positive | p value | |
| Diagnostic category | |||||||||
| Normal | 44 (47) | 49 (53) | 0.045 | 44 (48) | 47 (52) | 0.012 | 74 (75) | 25 (25) | <0.001 |
| Low-grade CIN | 44 (51) | 42 (49) | 35 (39) | 54 (61) | 58 (70) | 25 (30) | |||
| High-grade CIN | 147 (54) | 123 (46) | 148 (55) | 121 (45) | 134 (48) | 148 (52) | |||
| Cancer | 43 (39) | 68 (61) | 46 (40) | 69 (60) | 51 (42) | 71 (58) | |||
| Age, years | |||||||||
| <50 | 23 (34) | 44 (66) | 0.32 | 24 (34) | 46 (66) | 0.13 | 30 (40) | 46 (60) | 0.57 |
| >50 | 20 (46) | 24 (54) | 22 (49) | 23 (51) | 21 (46) | 25 (54) | |||
| FIGO stage | |||||||||
| I | 41 (41) | 59 (59) | 0.20 | 41 (40) | 62 (60) | 0.90 | 47 (43) | 63 (57) | 0.76 |
| II | 2 (18) | 9 (82) | 5 (42) | 7 (58) | 4 (33) | 8 (67) | |||
| Differentiation | |||||||||
| Well/moderate | 30 (41) | 44 (59) | 1.00 | 30 (40) | 46 (60) | 0.67 | 35 (44) | 44 (56) | 1.00 |
| Poor | 13 (42) | 18 (58) | 15 (46) | 18 (54) | 16 (44) | 20 (56) | |||
| Cell type | |||||||||
| SCC | 34 (38) | 56 (62) | 0.43 | 37 (40) | 56 (60) | 1.00 | 39 (39) | 60 (61) | 0.22 |
| AD | 9 (50) | 9 (50) | 8 (42) | 11 (58) | 11 (55) | 9 (45) | |||
| Tumor size | |||||||||
| <4 cm | 31 (37) | 54 (63) | 0.49 | 28 (32) | 59 (68) | 0.004 | 34 (36) | 60 (64) | 0.029 |
| ≥4 cm | 12 (46) | 14 (54) | 18 (64) | 10 (36) | 17 (61) | 11 (39) | |||
| LVI | |||||||||
| Negative | 24 (43) | 32 (57) | 0.67 | 20 (35) | 37 (65) | 1.00 | 25 (39) | 39 (61) | 0.83 |
| Positive | 12 (38) | 20 (62) | 13 (37) | 22 (63) | 15 (43) | 20 (57) | |||
| LN metastasis | |||||||||
| Negative | 36 (39) | 57 (61) | 0.78 | 39 (41) | 56 (59) | 0.43 | 42 (41) | 60 (59) | 0.80 |
| Positive | 7 (44) | 9 (56) | 5 (28) | 13 (72) | 8 (44) | 10 (56) | |||
Significant differences are highlighted in bold font. AD = Adenocarcinoma; LN = lymph node; LVI = lymphovascular invasion; SCC = squamous-cell carcinoma.
Prognostic Significance of AMPK Expression
The 5-year survival rate in the AMPKα2 overexpression group was 92.2%; the corresponding rate in the lower AMPKα2 expression group was 72.1% (fig. 4 b). AMPKα2 overexpression showed longer survival by univariate (HR 0.29, 95% CI 0.10–0.86, p = 0.026) analysis. AMPKα2 showed a trend toward favorable prognostic significance in multivariate analysis (HR 0.36, p = 0.08; table 3), without statistical significance.
Fig. 4.
a–e Kaplan-Meier survival curves according to AMPKα1, AMPKα2 and p-AMPKα expression. High AMPKα2 expression was associated with favorable DFS (b; p = 0.017). Combined high AMPKα2/p-AMPKα overexpression tended to be associated with longer DFS than that in patients with low AMPKα2 and p-AMPKα coexpression (e). There was no significant difference in survival according to the expression of AMPKα1 (a) or p-AMPKα (c). p values were obtained from log-rank tests.
Table 3.
Univariate and multivariate analyses of the association between prognostic variables and DFS in 104 cases of cervical cancer
| Risk factor | Univariate |
Multivariate |
||
|---|---|---|---|---|
| HR (95% CI) | p value | HR (95% CI) | p value | |
| FIGO stage II | 4.30 (1.35–13.71) | 0.014 | 4.46 (1.26–15.75) | 0.02 |
| Cell type, AD vs. SCC | 2.39 (0.75–7.64) | 0.14 | 3.52 (1.00–12.33) | 0.049 |
| Tumor size, ≥4 cm | 3.65 (1.28–10.42) | 0.015 | 2.09 (0.68–6.41) | 0.20 |
| AMPKα1 | 1.21 (0.37–4.03) | 0.75 | 1.36 (0.0.39–4.79) | 0.63 |
| AMPKα2 | 0.29 (0.10–0.86) | 0.026 | 0.36 (0.11–1.14) | 0.08 |
| p-AMPKα | 0.99 (0.32–3.03) | 0.99 | 1.62 (0.46–5.75) | 0.46 |
Significant differences are highlighted in bold font.
DFS of cervical cancer patients with both AMPKα2 and p-AMPKα overexpression (90.5%) tended to be longer than that of patients without AMPKα2 and p-AMPKα coexpression (79.2%, p = 0.23; fig. 4 e), but without statistical significance. In addition, there was no significant difference in survival according to the expression of AMPKα1 and p-AMPKα.
Discussion
We examined the expression of AMPKα1, AMPKα2 and p-AMPKα and its clinical significance in cervical cancer patients. p-AMPKα expression increased gradually in cancer tissues during cervical carcinogenesis, without prognostic significance. Notably, the AMPKα2 isoform showed potential as a favorable prognostic marker in cervical cancer. Further validation or additional study cohorts are required. This result suggested that isoforms and phosphorylated forms of AMPK play different roles in cervical carcinogenesis and prognosis.
Previous studies have identified AMPK as a potential metabolic tumor suppressor, and a loss of AMPK protein or activity in tumor cells allows for uncontrolled growth under physiological stress, such as the nutrient or oxygen deprivation often associated with tumor microenvironments [5, 24, 25]. In our study, the expression of AMPKα2 and p-AMPKα was associated with small-sized tumors, which is consistent with the role of AMPKs in growth control. However, expression of the α1 isoform did not show any correlation with tumor size.
In support of this observation, a previous study showed that AMPKα2-null H-RasV12-transformed cells demonstrate a significant anchorage-independent growth advantage [26]. Although a similar growth advantage was seen in AMPKα1-null H-RasV12 mouse embryonic fibroblasts, it was suggested that the compensatory increase in expression of AMPKα2 could also be driving these cells into apoptosis and preventing cellular transformation altogether. They concluded that the loss of AMPKα1 or AMPKα2 promotes cell growth via increases in MAPK pathway signaling, but that only the loss of AMPKα2 is permissive for overall growth and survival advantage of normal mouse embryonic fibroblasts [26].
Mutation of the MAPK pathway is one of the most common mutations found in cervical cancer. Recently, Ojesina et al. [7] performed whole-exome, transcriptome and whole-genome sequencing in cervical carcinomas. The results showed recurrent E322K substitutions in the MAPK1 gene (8%) in 79 primary squamous-cell carcinomas. In addition, several investigators have shown that AMPK likely modulates MAPK pathways through targeting of C-Raf directly or indirectly through KSR2, which would be reduced by the loss of AMPKα1 or AMPKα2 [27, 28].
AMPKα2 was associated with favorable prognosis in this study. This is in agreement with results from several previous studies that showed low AMPK expression in patients with worse survival outcomes and higher-grade tumors [17, 18, 29]. These studies suggest that the activation of AMPK regulates cell proliferation and apoptosis by p53, and inhibits cell growth and protein synthesis by inactivating mTOR [5, 30]. Molecular signaling pathways are implicated in cervical cancer development including PI3K/AKT, RAF/MEK/ERK and Wnt/β-catenin signaling. Recent studies demonstrated that there is a complex interrelationship between mitogenic signaling and LKB1/AMPK signaling [31]. Specifically, overexpression of E6 and E7 proteins from high-risk HPV is the main oncogenic stimulus for cell transformation in cervical cancer [32]. The early proteins, E6 and E7, upregulate Akt activity [33]. Akt activates mTOR by regulating the cellular ATP level and AMPK activity [34]. With these results, high-risk HPV regulates AMPK activity through Akt during cervical cancer progression.
Although previous studies have focused on the role of AMPK as a suppressor of cell growth, recent studies have begun to elucidate how the energy-sensing function of AMPK can promote cell survival [35, 36]. Mandal et al. [37] have shown that AMPK may act as a conditional tumor suppressor or oncogene, depending on the degree of AMPK activation, the particular AMPK isoforms present and other processes activated in the cell. Also, the necessity for AMPK in oncogenic transformation was suggested, via its role as a master sensor and regulator of cellular energy homeostasis, prerequisites for cancer cell survival [38]. Several factors may contribute to this contradictory significance, such as different types of cancer and different stages of the disease. In this study, the phosphorylated form of AMPK expression increased gradually during cancer progression.
In conclusion, p-AMPKα was associated with cervical carcinogenesis, and high expression of AMPKα2 was correlated with better DFS in patients with early-stage cervical cancer. However, we were unable to evaluate the prognostic significance of AMPKα2 expression for predicting progression to invasive cancer because most of the CIN II and CIN III patients enrolled in the study did not have recurrence or progression of cancer after treatment. This poses a challenge to further clarify the complex contribution of the AMPK isoforms and of phosphorylation status to the progression and prognosis of cervical cancer.
Supplementary Material
Acknowledgements
This research was supported by the Intramural Research Program of the National Institutes of Health National Cancer Institute, Center for Cancer Research.
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