Nucleophosmin overexpression is associated with poor survival in ...

APMIS 123: 515–522

© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12381

Nucleophosmin overexpression is associated with poor survival in astrocytoma YEN-HSIN KUO,1,2,† YI-TING CHEN,1,3,† HUNG-PEI TSAI,1 CHEE-YIN CHAI1,2,3,4,† and AIJ-LIE KWAN5,6,7,† 1

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung; Department of Pathology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung; 3Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung; 4Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung; 5 Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung; 6Department of Surgery, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; and 7 Department of Neurosurgery, University of Virginia, Charlottesville, VA, USA 2

Kuo Y-H, Chen Y-T, Tsai H-P, Chai C-Y, Kwan A-L. Nucleophosmin overexpression is associated with poor survival in astrocytoma. APMIS 2015; 123: 515–522. The multiple functions of the protein nucleophosmin (NPM) include the regulation and balance of cell growth, proliferation, and apoptosis. Many cancers have suspected associations with overexpression of NPM or with mutation of the NPM gene. Although NPM and anaplastic lymphoma kinase fusion proteins are known to be related to the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) signaling pathway, the relationships of NPM, JAK2, and STAT5 to astrocytoma remain unclear. Therefore, this study performed histochemical analyses of expressions of NPM, p-JAK2, and STAT5B proteins in patients with astrocytoma. The results showed that high NPM expression was significantly associated with high tumor grade (p = 0.000), old age (p = 0.000), low Karnofsky Performance Scale (KPS) score (p = 0.000), and tumor recurrence (p = 0.045). High p-JAK2 expression was significantly associated with old age (p = 0.000), high tumor grade (p = 0.000), low KPS score (p = 0.000), and tumor recurrence (p = 0.036). Expression of STAT5B was significantly correlated with tumor grade (p = 0.018) and KPS score (p = 0.002). High expressions of NPM, p-JAK2, and STAT5B were associated with a short survival time (p = 0.035, 0.003 and 0.002, respectively). In multivariable analysis, STAT5B expression was a significant predictor of survival time (p = 0.003). In conclusion, NPM and p-JAK2/STAT5B may have important roles in tumor progression, and STAT5B is an independent prognostic marker of astrocytoma. Key words: Nucleophosmin; JAK/STAT pathway; immunohistochemistry; astrocytoma. Chee-Yin Chai, Department of Pathology, Kaohsiung Medical University Hospital, No.100, Tzyou 1st Road, Kaohsiung 807, Taiwan. e-mail: [email protected] Aij-Lie Kwan, Department of Neurosurgery, Kaohsiung Medical University Hospital, No.100, Tzyou 1st Road, Kaohsiung 807, Taiwan. e-mail: [email protected] †

These authors contributed equally to this work.

Gliomas, the most common group of primary brain tumors, include astrocytoma, ependymoma and oligodendroglioma. Astrocytic tumors include noninfiltrating and infiltrating astrocytomas, the latter of which are implicated in approximately 60% of primary brain tumors in adults. The World Health Organization (WHO) grading scheme for astrocy-

Received 11 June 2014. Accepted 3 February 2015

toma is used to predict its biological behavior and clinical outcomes (1). Pilocytic astrocytoma typically occurs in children or young adults. Because of their biologically slow growth, these infratentorial lesions are associated with long survival. In contrast, according to the WHO classification scheme for astrocytoma, grade II (low grade), and grades III–IV (high grade) are malignant infiltrating tumors with poorer prognosis proportional to histological grade, i.e., diffuse astrocytoma (WHO grade 515

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II), anaplastic astrocytoma (AA) (WHO grade III), and glioblastoma multiforme (GBM) (WHO grade IV). Malignant astrocytomas are generally located in the supratentorial area with increased cellularity and cellular atypia. Histopathologically, AA is characterized by substantial nuclear pleomorphism with significant proliferative activity, and GBM is characterized by microvascular proliferation or tumor necrosis. Of the tumors in this category, GBM, which is often unresectable, is the most aggressive and has the poorest prognosis after combined chemoradiotherapy. Although clinicopathological parameters have been established to predict outcomes of astrocytoma, the mechanisms of tumor progression are still unclear. Nucleophosmin (NPM) is a phosphorylated protein located in the granular region of the nucleolus. Its multiple functions include regulating and balancing cell-growth, proliferation and apoptosis (2). The high oncogenic potential of NPM is indicated by reports of mutation or overexpression of NPM in many hematologic and solid organ malignancies (3–6). A proteomics analysis of high-grade astrocytoma by Gimenez et al. (7) also revealed up-regulated NPM protein level. The binding of extracellular ligands to various membrane receptors activates phosphorylation of signal transducer and activator of transcription (STAT) proteins by Janus protein tyrosine kinase (JAK) (8, 9). The STATs are known to have latent cytoplasmic transcription factors that respond to extracellular signaling proteins, especially cytokines, hormones, interferon, and growth factors (10, 11). This pathway has an important role in cell inflammation, proliferation, differentiation, migration, and apoptosis. Overexpression or chromosomal translocation of JAK/STAT proteins has been associated with hematologic diseases and other malignancies. Both JAK2 and STAT5 are also believed to be highly active in GBM cell lines (12). Studies indicate that NPM and anaplastic lymphoma kinase (ALK) fusion protein have roles in the JAK/STAT signaling pathway. Phosphorylation of STAT5B activates JAK-2, which decreases insulin growth factor (IGF). In anaplastic large cell lymphoma, NPM may moderate cell survival and invasion through JAK-2 and STAT5B (13). However, the relationship between NPM and JAK2/STAT5B and their clinical significance in human astrocytoma is still unclear. Therefore, this study performed immunohistochemical (IHC) analyses to identify the expressions of these three proteins and their clinicopathologic parameters in 99 astrocytoma patients.

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MATERIALS AND METHODS Tissue samples This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-20120071). The analysis included 99 cases of astrocytoma diagnosed from 1998 to 2011. In all cases, slides were reviewed to confirm the tumor differentiation grade based on the World Health Organization (WHO) classification system.

Immunohistochemical staining The specimens were fixed in 10% formalin and then embedded in paraffin. A microtome was used to cut the paraffin-embedded tissues into thicknesses of 3 lm. After using streptavidin-biotin method to detect NPM, p-JAK2, and STAT5B by IHC analysis, specimens were deparaffinized, rehydrated, and treated with 0.1 M citrate buffer (pH 6.0) in an autoclave at 121 °C for 8 min. Endogenous peroxidase activity was blocked by 5 min treatment with 3% hydrogen peroxide at room temperature. After washing with Tris buffer solution (TBS), the sections were incubated with the primary antibodies of NPM (1:200; Santa Cruz Biotech, Santa Cruz, CA, USA), p-JAK2 (1:50; Santa Cruz Biotech), and STAT5B (1:100; Santa Cruz Biotech) at room temperature for 1 h. The REAL Envision Detection System (Dako, Glostrup, Denmark) was performed by applying biotinylated secondary antibody and peroxidase-conjugated streptavidin for 30 min each. Finally, sections were incubated in 3,30 -diaminobenzidine for 5 min, counterstained with hematoxylin, and mounted. Cervical cancer, breast cancer, and colon carcinoma tissues were used as positive controls for IHC staining of NPM, p-JAK2, and STAT5B, respectively.

Scoring Two pathologists independently performed semi-quantitative analyses of expressions of NPM, p-JAK2, and STAT5B. The NPM protein was present in nuclei, and p-JAK2 and STAT5B proteins were present in both nuclei and cytoplasm. The numbers of positive-stained cells was estimated using three representative microscopic fields. Percentages of nuclear immunoreactivity of NPM in tumor cells were scored as ≦ 50% (low expression) or as >50% (high expression). The p-JAK2 and STAT5B were scored according to two characteristics: overall stain intensity and percentage of positively stained neoplastic tissue. The staining intensity value ranged from 0 to 3. Percentages of immunoreactivity in tumor cells were scored as follows: 1, 0–10%; 2, 11–75%; 3, 76–100%. Finally, expressions of p-JAK2 and STAT5B were categorized as low if the staining intensity value multiplied by the immunoreactivity score was ≦ 3 and high if the staining intensity value multiplied by the immunoreactivity score was >3.

Statistical analysis Statistical analyses were performed using SPSS version 14.0 for Windows (Chicago, IL, USA). The X2 test was

© 2015 APMIS. Published by John Wiley & Sons Ltd

NUCLEOPHOSMIN IN ASTROCYTOMA

performed to analyze the relationship between protein expression and clinical characteristics. The Pearson correlation coefficient was used to analyze NPM in terms of correlations with p-JAK2 and STAT5B. Overall survival was evaluated by the Kaplan–Meier method. Multivariable Cox proportional hazards regression models were used to assess the association between survival and clinicopathologic characteristics after adjusting for other factors. A p value < 0.05 was considered statistically significant.

grade II astrocytomas, 72% had low NPM expression, and the remaining 28% had high NPM expression. High-grade astrocytomas, including grades III and IV, had much higher NPM immunoreactivity (73.3%). High NPM expression was significantly associated with short survival time in Kaplan–Meier method (p = 0.035) (Fig. 2), but not in multivariable analysis.

RESULTS

Immunohistochemical expression and cellular distribution of p-JAK2 in human astrocytoma

Immunohistochemical analyses of NPM expression and cellular distribution in human astrocytoma

Figure 1 shows that the nuclei of tumor cells in human astrocytomas exhibited NPM immunoreactivity. Table 1 compares NPM expression in different astrocytoma types and in different patient characteristics. Of the 99 tumors analyzed, NPM expression was low in 48 (48.5%) and high in 51 (51.5%). Chi-square test showed that NPM expression was significantly associated with age (p = 0.000), tumor grade (p = 0.000), KPS (p = 0.000), and tumor recurrence (p = 0.045). All grade I astrocytomas had low NPM expression. In the

Figure 1 shows that the nuclear and cytoplasmic compartments of tumor cells in the human astrocytomas showed p-JAK2 immunoreactivity. Table 2 compares p-JAK2 expression in different astrocytoma types and in different patient characteristics. Of the 99 tumors analyzed, p-JAK2 expression was low in 66 (66.6%) and high in 33 (33.4%). Chisquare test showed that p-JAK2 expression was significantly associated with age (p = 0.000), tumor grade (p = 0.000), KPS (p = 0.000), and recurrence (p = 0.036). Patients with high p-JAK2 expression tended to be characterized by advanced age, high tumor grade, low KPS, and tumor recurrence. All

Fig. 1. Immunohistochemical staining results for NPM, p-JAK2, and STAT5B in low- and high-grade astrocytomas. Overexpressions of NPM, p-JAK2, and STAT5B were noted in high-grade astrocytoma (original magnification 9200). © 2015 APMIS. Published by John Wiley & Sons Ltd

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Table 1. Expression of NPM for varying clinicopathologic parameters of astrocytoma Parameters No. patients (n) NPM expression (n, %) Low Age ≦45 48 32 (66.7) >45 51 16 (31.4) Gender Male 51 25 (49.0) Female 48 23 (47.9) Tumor grade I 14 14 (100.0) II 25 18 (72.0) III 16 4 (25.0) IV 44 12 (27.3) KPS ≦70 67 24 (35.8) >70 32 24 (75.0) Recurrence No 65 36 (55.4) Yes 34 12 (35.3) Tumor size ≦3 cm 87 44 (50.6) >3 cm 12 4 (33.3) *Statistically significant (p < 0.05).

grade I astrocytomas showed negative or low pJAK2 expression while more than half of the grade III and grade IV astrocytomas had high immunoreactivity. Figure 2 shows that p-JAK2 expression was significantly (p = 0.003) associated with short survival time in Kaplan–Meier method but not in multivariable analysis. Immunohistochemical expression and cellular distribution of STAT5B in human astrocytoma

Figure 1 shows the positive immunostaining results for STAT5B immunoreactivity observed in the cytoplasm and nuclei of tumor cells. Table 3 compares STAT5B expression in different astrocytomas and in different patient characteristics. Of the 99 tumors analyzed, STAT5B expression was low in 49 (49.5%) and high in 50 (50.5%). Chi-square test indicated that STAT5B expression significantly correlated with tumor grade (p = 0.018) and with KPS (p = 0.002). Most (71.4%) grade I astrocytomas showed low immunostaining, but more than half (54.1%) of the malignant astrocytomas had high immunostaining. High immunoreactivity was observed more frequently in high-grade astrocytomas (63.3%) than in low-grade astrocytomas (30.8%). Figure 2 shows the Kaplan–Meier results, which revealed a significant (p = 0.002) association between low STAT5B expression and long-term survival time. Multivariable analysis revealed that, STAT5B expression was significantly associated with overall survival (p = 0.003). 518

p value* High 16 (33.3) 35 (68.6)

0.000

26 (51.0) 25 (52.1)

0.536

0 7 (28.0) 12 (75.0) 32 (72.7)

0.000

43 (64.2) 8 (25.0)

0.000

29 (44.6) 22 (64.7)

0.045

43 (49.4) 8 (66.7)

0.179

Associations among NPM, p-JAK2 and STAT5B

Pearson correlation coefficients were computed to assess relationships among NPM, p-JAK2 and STAT5B. Expression of NPM correlated positively with expression of p-JAK2 (r = 0.300, n = 99, p = 0.003) and with expression of STAT5B (r = 0.314, n = 99, p = 0.002). Overall, increased expression of NPM correlated with increased expressions of p-JAK2 or STAT5B. However, p-JAK2 expression did not correlate with STAT5B expression. DISCUSSION Biologically, astrocytes in the central neuron system are responsible for providing the nutrition needed to support formation of nervous system tissue, formation of the blood–brain barrier, and maintenance of the extracellular microenvironment. Once oncogenesis occurs, patients may suffer from neurogenic symptoms, the combined effects of which may even cause death. Low-grade astrocytomas generally have slow growth, and their average survival time is more than 5 years. In contrast, high-grade astrocytomas have faster growth, more aggressive behavior, shorter survival time, and earlier recurrence. The overall survival rate for GBM was 40% in the first year and did not substantially improve after combined chemotherapy and radiotherapy (1). Astrocytic neoplasms are morphologically classified according to the WHO grading system. However,



A

Fig. 2. Kaplan–Meier survival curves for (A) NPM, (B) p-JAK2, and (C) STAT5B. High expressions of NPM, p-JAK2, and STAT5B were significantly associated with short-term survival time (p < 0.05).

B

C


standard histological methods cannot accurately predict tumor progression in such patients. Therefore, their pathogenesis and molecular markers require careful study to provide guidelines for effective treatment modalities. The NPM, also known as nucleolar phosphoprotein B23, is a multifunctional protein encoded by the NPM gene. The many roles of NPM include ribosome genesis, protein chaperoning, centrosome duplication, histone assembly and cell proliferation (2). It also maintains genomic stability and regulates alternative reading frame (ARF) proteins via the p53 tumor suppressor pathway (14). The NPM is expressed at physiological levels and has a major role in balancing cell proliferation with cell apoptosis. The oncogenic potential of NPM overexpression in tumor cells includes programmed cell death and tumor growth, division, and differentiation (15). Chromosomal translocation of the NPM gene is a well-known cause of hematologic neoplasms such as anaplastic large cell lymphoma, myelodysplastic syndrome, and acute promyelocytic leukemia (9). Overexpression of NPM proteins has also been reported in many solid organ cancers. Proteomics studies indicate that NPM proteins are more abundant in high-grade astrocytomas than in low-grade or non-neoplastic tissues (7). Similarly, the current study revealed that high NPM expression was associated with high tumor grade and short overall survival time. The JAK/STAT pathway has a central role in cellular regulation. During signal transduction, JAKs are activated and phosphorylated by ligands binding to cell surface receptors. The binding of phosphorylated JAKs to STATs then dimerizes phosphorylated STAT protein and translocates signals into the cell nucleus. The resulting transcription factors interact with specific DNA-binding sites such as Bcl-xl, c-myc, interleukin (IL)-10, and PRAD-1 gene (16). Therefore, the JAK/STAT pathway contributes to many diseases, especially cancers. The STAT5B is a transcription factor that contributes to cancer and to immune and growth disorders (17, 18). Studies show that the NPMALK signaling pathways mediate cell cycles through JAK-STAT and PI3K-AKT signaling pathway and regulated by BCL-xl and FOXO3A (19, 20). Another important function of JAKs is

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Table 2. Expression of p-JAK2 for varying clinicopathologic parameters of astrocytoma Parameters No. patients (n) p-JAK2 expression (n, %) Low High Age ≦45 48 41 (85.4) 7 (14.6) >45 51 25 (49) 26 (51.0) Gender Male 51 38 (74.5) 13 (25.5) Female 48 28 (58.3) 20 (41.7) Tumor grade I 14 14 (100) 0 II 25 23 (92) 2 (8) III 16 7 (43.75) 9 (56.2) IV 44 22 (50.0) 22 (50.0) KPS ≦70 67 34 (50.7) 33 (49.3) >70 32 32 (100) 0 Recurrence No 65 48 (73.9) 17 (21.5) Yes 34 18 (52.9) 16 (47.1) Tumor size ≦3 cm 87 59 (67.8) 28 (32.2) >3 cm 12 7 (58.3) 5 (41.7) *Statistically significant (p < 0.05).

Table 3. Expression of STAT5B for varying clinicopathologic parameters of astrocytomas Parameters No. patients (n) STAT5B expression (n, %) Low High Age ≦45 48 26 (54.2) 22 (45.8) >45 51 23 (45.1) 28 (54.9) Gender Male 51 24 (47.1) 27 (52.9) Female 48 25 (52.1) 23 (47.9) Tumor grade I 14 10 (71.4) 4 (28.6) II 25 17 (68) 8 (32) III 16 6 (37.5) 10 (62.5) IV 44 16 (36.4) 28 (63.6) KPS ≦70 67 26 (38.8) 41 (61.2) >70 32 23 (71.9) 9 (28.1) Recurrence No 65 30 (46.2) 35 (53.8) Yes 34 19 (55.9) 15 (44.1) Tumor size ≦3 cm 87 43 (49.5) 44 (50.6) >3 cm 12 6 (50) 6 (50) *Statistically significant (p < 0.05).

mediation of cytokine inflammation and STAT function (8, 9). By activating signaling pathways and by phosphorylating tyrosine, JAKs promote translocation from cytoplasm to the nucleus by phosphorylation to STAT5 (21). The NPM and ALK fusion protein are related to the JAK/STAT5 signaling pathway in malignant transformation of lymphoid cells. Additionally, JAK2 is constitutively tyrosine-phosphorylated in anaplastic large cell 520

p value*

0.000 0.088 0.000

0.000 0.036 0.514

p value*

0.367 0.617 0.018

0.002 0.358 0.970

lymphoma cells and in NPM/ALK-transformed hematopoietic cells, which enhances cell proliferation and resistance to apoptosis. The STAT5B, which requires JAK2, regulates NPM transformation (22). In vivo and in vitro studies show that inhibition of STAT5B impairs NPM-ALK cell transformation by triggering apoptosis (23). In anaplastic large cell lymphoma, NPM proteins also moderate cell survival and invasion through the © 2015 APMIS. Published by John Wiley & Sons Ltd


JAK2/STAT5B pathway (13). In the central neuron system, JAK2 promotes axon growth by mediating cytokine suppression by STAT5 (24). Members of the JAK/STAT family of proteins present in the embryonic and postnatal brain regulate astroglia genesis during maturation (25). Inhibition of STAT5B may suppresses proliferation and reduce tumor cell invasion in GBM (26). Our study revealed that high p-JAK2 expression was associated with old age, high-grade tumor formation, low KPS, and tumor recurrence. Similarly, STAT5B expression was associated with high tumor grade and low KPS. Patients with low expression of p-JAK2 or STAT5B had improved long-term survival. The NPM also revealed a positive correlation with p-JAK2 and STAT5B. The data suggest that both p-JAK2 and STAT5B contribute to tumor progression in astrocytoma and have potential use as prognostic markers of patient outcome.

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CONCLUSION This study used IHC staining to investigate the relationships among expressions of NPM, p-JAK2, and STAT5B and their significant associations with clinical outcome. The results showed that expressions of the NPM, p-JAK2, and STAT5B proteins correlated with tumor grade and survival time in patients with astrocytoma. In multivariable analysis, STAT5B expression was significantly associated with overall survival. Additionally, NPM, p-JAK2 and STAT5B protein expressions were low in low-grade astrocytoma (grades I–II) and high in more than 50% of tumor cells in high-grade astrocytoma (grades III– IV). Additionally, NPM was significantly associated with JAK2/STAT5B. In conclusion, NPM and pJAK2/STAT5B may have important roles in tumor progression, and STAT5B can be considered an independent prognostic marker of astrocytoma. However, further studies are needed to clarify their precise role in tumor pathogenesis. REFERENCES 1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. WHO Classification of Tumours of the Central Nervous System. Lyon: IARC, 2007. 2. Lindstrom MS. NPM1/B23: a multifunctional chaperone in ribosome biogenesis and chromatin remodeling. Biochem Res Int 2011;2011:195209. 3. Tanaka M, Sasaki H, Kino I, Sugimura T, Terada M. Genes preferentially expressed in embryo stomach are predominantly expressed in gastric cancer. Cancer Res 1992;52:3372–7. 4. Nozawa Y, Van Belzen N, Van der Made AC, Dinjens WN, Bosman FT. Expression of nucleophos-


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