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Clinicopathological significance of BGP expression in non-small-cell lung carcinoma: relationship with histological type, microvessel density and patients’ survival
Pathology (December 2006) 38(6), pp. 555–560
ANATOMICAL PATHOLOGY
Clinicopathological significance of BGP expression in non-small-cell lung carcinoma: relationship with histological type, microvessel density and patients’ survival MIN KI LEE*{, JAE HO KIM{, CHANG HUN LEE*§, JONG MIN KIM**, CHI DUK KANGd, YOUNG DAE KIM", KYUNG UN CHOI§, HWAL WOONG KIM§, JEE YEON KIM§, DO YOUN PARK§ AND MEE YOUNG SOL§ *Medical Research Institute, and Departments of {Internal Medicine, {Physiology, §Pathology, dBiochemistry and "Thoracic Surgery, College of Medicine, Pusan National University, Busan, Korea; **Department of Anatomy, College of Medicine, Dong A University, Busan, Korea
Summary Aims: Brain-type glycogen phosphorylase (BGP) is the major isoform of glycogen phosphorylase found in fetal and neoplastic tissues, and is generally thought to induce glucose supply during an ischaemic period. This study was performed to investigate BGP expression in non-small-cell lung carcinoma (NSCLC). Methods: A total of 119 cases of NSCLC, including 63 squamous cell carcinomas (SqCCs) and 56 adenocarcinomas (ACs), were imunohistochemically evaluated for BGP expression, and its expression was correlated with clinicopathological parameters. Results: In total, 76.5% were positive, while non-neoplastic bronchial epithelial cells were weakly positive and pneumocytes were negative. High BGP expression was noted in 78.6% of ACs and 36.5% of SqCCs (p50.001). Microvessel density was higher in the low BGP expression tumours (29.6 ¡ 16.9/mm2) than in the high expression tumours (22.8 ¡ 13.8/mm2) (p50.017). BGP expression did not correlate with patient age or tumour stage, but was more frequent in females than males. Kaplan–Meier analysis showed that high BGP expression was associated with poorer survival (p50.032). Conclusions: BGP is expressed in NSCLC, particularly AC, and is an independent poor prognostic factor. Key words: Brain-type glycogen phosphorylase, BGP, non-small-cell lung carcinoma, NSCLC, histology, microvessel density, survival. Received 4 June, revised 18 August, 5 September, accepted 11 September 2006
INTRODUCTION In epithelial cells of the developing respiratory tract, mobilisation of glycogen stores is regulated differentially such that glycogenolysis in the alveolar epithelium generally precedes that in the bronchial and bronchiolar epithelium.1 Glycogen phosphorylase (GP) catalyses the phosphorolysis of glycogen to glucose 1-phosphate, which represents the initial step for the synthesis of pulmonary surfactant phospholipids in alveolar epithelium of the developing airway. Mammalian GPs are found in three major isozymes, i.e., muscle, liver and
brain, that can be distinguished by functional and structural properties as well as by the tissues in which they are predominantly expressed.2–4 cDNA sequences encoding the three human GP isoforms have been aligned.3,5 Although they have very similar enzymatic activities and primary structures (80–90% homology at amino acid level), chromosomal mapping analyses have revealed that the genes encoding muscle, liver, and brain-type GP (BGP) are assigned to chromosomes 11, 14, and 20, respectively.3 The physiological role of BGP is generally thought to be the provision of an emergency glucose supply during a stressful and ischaemic period, as may occur in neoplasms. Of note, the major isoform of GP found in fetal and tumour tissues is BGP.6,7 Most reported studies of BGP expression in human cancers focus on gastrointestinal tract carcinoma.8–10 In colorectal carcinoma, the immunohistochemical expression of BGP has been reported to be common, with significant increase during the colorectal adenoma-carcinoma sequence. In particular, positive BGP staining in noncancerous colorectal mucosa (called BGP foci) was observed mainly around adenocarcinomas without an adenomatous component. These results led to the suggestion that the BGP focus might contribute to carcinogenesis.8 In gastric cancer, a strong correlation was observed between BGP expression and both intestinaltype carcinoma and intestinal metaplasia, whereas no positive staining was noted in the normal gastric epithelial cells.9,11 Compared with these gastrointestinal carcinomas, BGP expression has not previously been reported in nonsmall-lung carcinoma (NSCLC). This study is designed to investigate for the first time the immunohistochemical localisation of BGP in NSCLC, and then evaluate the relationship between BGP expression and clinicopathological factors of NSCLC.
MATERIALS AND METHODS Patients and clinicopathological data All patients with NSCLC who had undergone lobectomy or pneumonectomy at Pusan National University Hospital (PNUH), Korea, during the
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020601024029
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period January 1998 to December 2002 were considered. After exclusion of cases in which there was insufficient pathological material remaining for further study, a total of 119 cases were selected for this study. There were 96 men and 23 women in the study population. Mean age was 58 years (range 27–79 years). Tumours were staged according to the TNM classification of the International Union Against Cancer (UICC) staging system12 after reviewing the clinical, radiological, and pathological data. Other clinical information was extracted from medical records. For survival analysis, the follow-up information was also checked. Patient survival was calculated as the time between operation and death. Patients who were still alive at the time of data collection were censored in the statistical analysis. The surgically resected specimens were fixed immediately in 10% buffered formalin (pH 7.0). All sections containing both tumour tissue and surrounding lung tissue were embedded in paraffin, and five serial sections (4 mm) were cut from a selected, paraffin-embedded tissue block. For pathological diagnosis, one of these sections was stained with H&E. The others were used for immunohistochemistry. Pathological diagnosis was based on the third edition of the World Health Organization classification.13 The tumours included 63 cases of squamous cell carcinoma (SqCC) and 56 of adenocarcinoma (AC). Additionally, part of the fresh specimens from cancer foci and their surrounding lung tissues were frozen at 280uC for Western blot analysis. Immunohistochemistry Sections from paraffin-embedded blocks were transferred to poly-L-lysine coated glass slides and air-dried overnight at 37uC. They were dewaxed in xylene (three changes), rehydrated in a graded series of decreasing ethanol concentrations, and then rinsed in Tris-buffered saline (50 mM Tris/HCl, pH 7.4, containing 100 mM sodium chloride). Endogenous peroxidase activity was inactivated with 5% hydrogen peroxide in methanol for 15 min at 37uC. The following primary antibodies were used: anti-BGP (rabbit polyclonal anti-human BGP, 1:500), anti-CD34 (mouse monoclonal antiCD34, QBEnd/10, 1:50; NeoMarkers, USA), and anti-proliferating cell nuclear antigen (PCNA; mouse monoclonal anti-PCNA, PC10, 1:1 500; Sigma Chemical Co., USA). Anti-BGP antibody was a generous gift from Dr Michio Ogawa (Department of Surgery II, Kumamoto University School of Medicine, Japan). Each primary antibody was incubated with the tissue sections overnight at 4uC, and then immunohistochemical procedures were performed using the Histostain Plus kit (Zymed Laboratories, USA) using the standard streptavidin-biotin complex method. The reaction products were visualised by exposing sections to 3-amino9-ethylcarbazole. Nuclei were lightly counterstained for about 20 s with Mayer’s haematoxylin. Sections were then mounted in diluted malinol after the application of Universal Mount (Dako, USA). In each staining batch, appropriate positive control specimens were used. Tissue samples incubated with non-immune serum served as negative controls. Evaluation of immunohistochemical stainings All immunohistochemical evaluation was performed by two independent observers (CHL and MKL). Discordance between the observers was resolved by consensus using a multiheaded microscope. For BGP, immunostaining was semiquantitatively graded on a scale of 0 to 3+ according to area percentage of cells with positive cytoplasmic staining: 0 for absent or less than 10%; 1+ for 10–25%; 2+ for 26–50%; and 3+ for more than 50%. Grades 0 and 1 were regarded as low expression, and grades 2 and 3 as high expression. CD34 immunostaining was performed to evaluate tumour angiogenic activity expressed as microvessel density (MVD), and CD34-positive microvessels were assessed in the surrounding stroma of invasive tumour nests as described in the literature.14 Briefly, in four adjacent fields of vision in the most vascularised area, microvessels were counted at 6200 magnification using an Olympus microscope (BX 50; Olympus, Japan), and then MVD was expressed as the mean value of microvessels/mm2 for each case. For PCNA, we examined approximately 1000 tumour cells from each tumour tissue for nuclear positivity at high power (6400) after screening for areas of highest staining intensity at low power (6100). The labelling index for PCNA was
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expressed as the percentage of cells showing positive nuclear staining in each tumour. Western blot analysis for BGP expression in normal lung and tumour tissues NSCLC tumour tissues (10 cases of AC and 10 cases of SqCC) and their respective normal lung tissues were homogenised for 3 min on ice in lysis buffer (20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM PMSF, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 25 mMglycerol phosphate, 1% Triton X-100, pH 7.4). The homogenates were centrifuged at 10 000 g for 10 min, and the supernatants were used as the protein extracts of tissues. The protein concentrations of the extracts were determined using the Bio-Rad protein assay kit (Bio-Rad, USA). On 10% SDS-polyacrylamide gel, 60 mg of the extract was resolved and electrotransferred to nitrocellulose membrane. The membrane was blocked in 5% non-fat milk in TBST (20 mM TrisHCl, 137 mM NaCl, 0.1% Tween 20, pH 7.6) and incubated with anti-BGP antibody (rabbit polyclonal anti-BGP, 1:2000) for 2 h at room temperature. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000; Jackson ImmunoResearch Laboratories, USA) for 1 h at room temperature. Immunoreactive proteins were detected with the enhanced chemiluminescence detection system (Amersham Biosciences, UK). Statistical analysis The Pearson x2 and Student’s t tests were used to evaluate whether BGP expression correlated with clinicopathological parameters. The Mann– Whitney U test was also used for quantitative comparison of BGP expression between histological types of NSCLC after densitometric conversion of Western blot bands. For univariate survival analyses, the Kaplan–Meier method was applied, and the difference between the survival curves was analysed by the log-rank test. The Cox proportional hazard model was used to estimate the hazard ratio (HR) of independent factors influencing patient’s survival. All analyses were performed on a personal computer with the SPSS statistical package (Release 12.0.1; SPSS Inc., USA). p values less than 0.05 were regarded as statistically significant. All statistical tests were twosided.
RESULTS BGP expression in non-neoplastic lung and carcinoma tissues Bronchial epithelial cells, bronchial glands and bronchial smooth muscle showed weak positive immunoreactivity to anti-BGP antibody (Fig. 1A). Alveolar epithelial cells (pneumocytes), stromal fibroblasts and inflammatory cells were negative. Immunoreactivity to anti-BGP antibody was found in 76.5% (91/119) of NSCLC cases. In AC, carcinoma cells usually showed diffuse strong positivity (Fig. 1B). Nonneoplastic alveolar epithelial cells were negative, which contrasted with adjacent carcinoma showing distinctive positive reaction (Fig. 1C). In SqCC, immunoreactivity was generally more heterogeneous in distribution and intensity (Fig. 1D). When the cancers were divided into two groups according to the levels of BGP expression, 43.7% (52/119) of cancers showed low BGP expression and 56.3% (67/119) showed high expression. Relationship between BGP expression and clinicopathological parameters As shown in Table 1, histological type, MVD, and patient sex correlated with BGP expression. High expression of BGP was present in 78.6% (44/56) of AC, in contrast to 36.5% (23/63) of SqCC. The level of BGP expression was
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Fig. 1 (A) In non-neoplastic lung tissue, BGP expression is weakly positive in bronchial epithelial cells, but negative in alveolar epithelial cells (6100). (B) Adenocarcinoma showing diffuse BGP expression (6200). (C) Area of adenocarcinoma with strong BGP expression is sharply contrasted with non-neoplastic alveolar cells showing negative BGP reaction (6200). (D) Squamous cell carcinoma showing focal BGP expression (6200). (E,F) CD34-postive microvessels (E) in adenocarcinoma with low BGP expression and (F) in squamous cell carcinoma with high BGP expression (6200). (G,H) PCNA expression (G) in adenocarcinoma with low BGP expression and (H) in squamous cell carcinoma with high BGP expression (6200).
significantly higher in ACs than in SqCCs (p,0.001) using the x2 test. The degree of differentiation in both histological types was not associated with the level of BGP expression. MVD, assessed by CD34 immunoreactivity, revealed a significant difference between the low and high expression groups of BGP (29.6¡16.9 microvessels/mm2 in the former versus 22.8¡13.8 microvessels/mm2 in the latter, p50.017; Fig. 1E,F; Table 1) using the Student’s t test. The PCNA index of the low BGP expression group was slightly higher
than that of high BGP group, although this was not significant (p50.408) using the x2 test (Fig. 1G,H). Regarding patient sex, high expression of BGP was present in 78.3% (18/23) of tumours in female patients and in 51.0% (49/96) of male patients. The BGP expression of female patients was significantly higher than that of male patients using the x2 test (p50.018). Otherwise, the level of BGP expression in NSCLC did not correlate with patient age or stage using the x2 test (p.0.05, respectively; Table 1).
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TABLE 1 Relationship between BGP expression and clinicopathological parameters in non-small-cell lung carcinomas BGP expression
Parameters Histological type SqCC AC Age (years) (58* .58 Sex Female Male Stage I II III TNM-pT T1 T2 T3-4 TNM-pN N0 N1-2 MVD (/mm2){ PCNA (%){
Total no. cases
Low (n552)
High (n567)
63 56
40 12
23 44
54 65
23 29
31 36
23 96
5 47
18 49
56 19 44
27 11 14
29 8 30
23 68 28
11 32 9
12 36 19
68 51 119 119
32 20 29.6¡16.9 49.9¡21.3
36 31 22.8¡13.8 46.5¡22.6
p value ,0.001 0.825 0.018 0.103
0.489
0.393 0.017 0.408
*Mean age group to create groups. {Expressed as mean¡SD. SqCC, squamous cell carcinoma; AC, adenocarcinoma; MVD, microvessel density; PCNA, anti-proliferating cell nuclear antigen.
Quantitative comparison of BGP expression according to histological types In Western blot analyses using the extracts from 20 NSCLCs (10 cases of AC and 10 of SqCC) and corresponding normal lung tissues, anti-BGP antibody showed the formation of characteristic 107 kDa bands, consistent with BGP molecule, in all materials (Fig. 2). When densitometry (LabWorks Software 4.0; Ultra-Violet Products, UK) was applied for the calculation of relative density ratios for tumour to normal bands, the ratios of ACs were significantly higher than those of SqCCs using the Mann–Whitney U test (p50.001; Fig. 3).
Fig. 3 Box plots of the values of tumour-to-normal relative ratios after densitometric calculation of BGP-specific bands from Western blot analyses. SqCC, squamous cell carcinoma; AC, adenocarcinoma; NSCLC, non-small-cell lung carcinoma.
survival was defined as the interval in months between the day of surgical resection and date of either death or the last follow-up. An observation was censored at the last followup when the patients were alive or had died from a cause other than cancer. With regard to BGP expression, 26 of 47 patients with low expression were censored and 21 died (mean and median survival times, 66.9 and 77.0 months, respectively). Twenty of 63 patients with high expression were censored and 43 died (mean and median survival times, 44.9 and 25.0 months, respectively). When Kaplan–Meier’s univariate analysis was performed, the overall survival was associated with the level of BGP expression (p50.032 by log-rank test; Fig. 4), but not with histological type, MVD, and patient sex (p50.581, 0.180, and 0.603, respectively). A Cox proportional hazard regression model also identified BGP expression as a single independent prognostic factor (hazard ratio 1.748; 95%
Survival analysis During the course of this study (maximal follow-up 132 months; minimal follow-up 1 month; median follow-up 23 months), nine cases were lost to follow-up. The length of
Fig. 2 Western blot analysis using rabbit polyclonal anti-human antibody to BGP in non-small-cell lung carcinomas. A single band is observed at 107 kDa in all samples of normal and tumour tissues, although its density is rather varied to the types of samples. Lanes 1–5, adenocarcinoma; lanes 6– 10, squamous cell carcinoma; N, non-neoplastic lung tissue; T, tumour tissue.
Fig. 4 Overall survival curves of patients with non-small-cell lung carcinoma showing low expression (n547) and high expression (n563) of BGP.
BGP EXPRESSION IN NON-SMALL-CELL LUNG CARCINOMA
confidence interval 1.037–2.948; p50.036), after controlling for potential confounding factors such as histological type, MVD, sex, age, and PCNA index.
DISCUSSION Glycogen accumulates to significant levels in epithelial cells of the developing respiratory tract. Glucose, originating from glycogen, provides substrate for the synthesis of fatty acid and glycerol which, in turn, are incorporated into surfactant dipalmitoyl phosphatidylcholine.15 Rannels et al.16 suggested that BGP activity was clearly detected in isolated fetal type II pneumocytes of developing rat lung and might be involved in mobilisation of type II cell glycogen during late fetal lung development. Glucose uptake and metabolism are essential for proliferation and survival of cells, and are thought to be enhanced in actively proliferating cell systems such as developing fetal lung epithelium and lung carcinomas.17,18 Thus, glycogenolysis by BGP may have an important role in tumour cell growth. However, the clinicopathological significance of the glycolytic enzyme BGP in NSCLC has not been described so far in the English literature. In the present study, positive immunoreactivity to antiBGP antibody was found in 76.5% (91/119) of NSCLC cases and the level of BGP expression was significantly higher in ACs than in SqCCs (p,0.001). Western blot data also showed that lung AC tissues contained distinctively enhanced tissue levels of BGP, whereas SqCC tissues usually had slightly higher levels of BGP compared with those of the corresponding non-neoplastic lung tissues. In normal lung tissues, pneumocytes were negative for antiBGP antibody. Normal bronchiolar and bronchial cells frequently showed diffuse positivity for the antibody, although the intensity of staining was usually weaker than in carcinoma cells. Tashima et al.8 suggested that the expression of BGP in colorectal cancers may be indicative of oncofetal antigen reversion in cancer, like a-fetoprotein in hepatocellular carcinomas or carcinoembryonic antigen in gastrointestinal cancers. They found that BGP was expressed in the colonic adenomas with higher grades of dysplasia and cancers, and there was positive correlation between BGP expression and the adenoma-carcinoma sequence. We could not evaluate BGP expression in preneoplastic stages of AC, but we did find that the degree of histological differentiation had no relationship to BGP expression. Our immunohistochemical and Western blot expression data might be explained by oncofetal conversion as postulated in gastrointestinal carcinomas. The cellular expression of BGP in SqCC could be associated partly with oncogenic over-expression of the molecule, although its exact biological mechanism is still not determined. Previous studies of the prognostic role of angiogenesis in NSCLC have been contradictory, with some workers finding poorer outcomes in tumours with high levels of angiogenesis19–21 and others finding no correlation.22–24 In our study we could not find any association between MVD and overall survival of NSCLC patients. However, we did find that the MVD of the low BGP expression group was significantly higher than that of the high BGP expression group, suggesting that BGP expression is increased
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as a result of tumour hypoxia. It appears that angiogenesis and BGP expression are independent variables in NSCLC. In the present study with NSCLCs, the overall survival time of the high BGP expression group was significantly shorter than that of the low BGP expression group. A multivariate analysis also confirmed that the level of BGP expression was an independent prognostic factor. There was no relationship between stage and the level of BGP expression. There was significantly higher BGP expression in the tumours of female patients than in males. Given that BGP expression is higher in AC than in SqCC, the sex difference is most likely explained on the basis of the higher incidence of AC in females. Uncontrolled cell proliferation is the hallmark of malignant tumours. However, the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial.25–28 The role of glycogen metabolism in cell proliferation and tumour formation is not well studied, even though altered regulation of carbohydrate metabolism has long been associated with cancer cells.29 Schnier et al.30 showed that when A549 NSCLC cells expressing BGP were treated by the GP inhibitor CP-91,149, the cells accumulated glycogen and demonstrated growth inhibition. Unexpectedly, the present study found that the PCNA index of tumours from the low BGP expression group was slightly higher than that of the high BGP group, although the result did not reach significance. This suggests that the requirement for glucose to provide energy for cell growth is not the sole reason for BGP expression. Instead, BGP expression in NSCLC might represent oncofetal conversion or over-expression phenomenon during carcinogenesis. In summary, we have described the incidence of BGP expression in non-small-cell lung carcinoma and demonstrated its significant association with adenocarcinoma and poor survival outcome. Further study is necessary to elucidate the underlying pathobiology of this association and the role, if any, of BGP in lung carcinogenesis. ACKNOWLEDGEMENTS The authors thank Dr Michio Ogawa (Department of Surgery II, Kumamoto University School of Medicine, Japan) for kindly providing rabbit polyclonal anti-human BGP antibody in the present study. We also thank Young Jae Lee for excellent technical assistance. This study was supported by Medical Research Institute Grant (2004-40), Pusan National University, Busan, Korea. Address for correspondence: Dr C. H. Lee, Department of Pathology, College of Medicine, Pusan National University, 1-10 Ami-dong, Seo-gu, Busan 602-739, Korea. E-mail: [email protected]
References 1. Maniscalco WM, Wilson CM, Gross I, Gobran L, Rooney SA, Warshaw JB. Development of glycogen and phospholipid metabolism in fetal and newborn rat lung. Biochim Biophys Acta 1978; 530: 333–46. 2. Newgard CB, Hwang PK, Fletterick RJ. The family of glycogen phosphorylases: structure and function. Crit Rev Biochem Mol Biol 1989; 24: 69–99. 3. Newgard CB, Littman DR, van Genderen C, Smith M, Fletterick RJ. Human brain glycogen phosphorylase. Cloning, sequence analysis, chromosomal mapping, tissue expression, and comparison with the human liver and muscle isozymes. J Biol Chem 1988; 263: 3850–7.
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4. Shimada S, Maeno M, Akagi M, Hatayama I, Sato T, Sato K. Immunohistochemical detection of glycogen phosphorylase isoenzymes in rat and human tissues. Histochem J 1986; 18: 334–8. 5. Nakano K, Hwang PK, Fletterick RJ. Complete cDNA sequence for rabbit muscle glycogen phosphorylase. FEBS Lett 1986; 204: 283–7. 6. Sato K, Morris HP, Weinhouse S. Phosphorylase: a new isozyme in rat hepatic tumors and fetal liver. Science 1972; 178: 879–81. 7. Takashi M, Koshikawa T, Kurobe N, Kato K. Elevated concentrations of brain-type glycogen phosphorylase in renal cell carcinoma. Jpn J Cancer Res 1989; 80: 975–80. 8. Tashima S, Shimada S, Yamaguchi K, Tsuruta J, Ogawa M. Expression of brain-type glycogen phosphorylase is a potentially novel early biomarker in the carcinogenesis of human colorectal carcinomas. Am J Gastroenterol 2000; 95: 255–63. 9. Matsuzaki H, Shimada S, Uno K, Tsuruta J, Ogawa M. Novel subtyping of intestinal metaplasia in the human stomach: brain-type glycogen phosphorylase expression in the proliferative zone and its relationship with carcinogenesis. Am J Clin Pathol 1998; 109: 181–9. 10. Shimada S, Shiomori K, Honmyo U, Maeno M, Yagi Y, Ogawa M. BGP expression in gastric biopsies may predict the development of new lesions after local treatment for early gastric cancer. Gastric Cancer 2002; 5: 130–6. 11. Shimada S, Maeno M, Misumi A, Takano S, Akagi M. Antigen reversion of glycogen phosphorylase isoenzyme in carcinoma and proliferative zone of intestinal metaplasia of the human stomach. An immunohistochemical study. Gastroenterology 1987; 93: 35–40. 12. Sobin LH, Wittekind C. Lung. In: Sobin LH, Wittekind C (International Union Against Cancer [UICC]), editors. TNM Classification of Malignant Tumours. 5th ed. Baltimore: Wiley-Liss. 1997; 93–7. 13. Travis WD, Colby TV, Corrin B, et al. Histological Typing of Lung and Pleural Tumours. International Histological Classification of Tumours. 3rd ed. Berlin: Springer-Verlag. 14. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. N Engl J Med 1991; 324: 1–8. 15. Farrell PM, Bourbon JR. Fetal lung surfactant lipid synthesis from glycogen during organ culture. Biochim Biophys Acta 1986; 878: 159–67. 16. Rannels SR, Liu L, Weaver TE. Expression of glycogen phosphorylase isozymes in developing rat lung. Am J Physiol 1997; 273: L389–94. 17. Ito T, Noguchi Y, Udaka N, Kitamura H, Satoh S. Glucose transporter expression in developing fetal lungs and lung neoplasms. Histol Histopathol 1999; 14: 895–904.
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18. Roach PJ. Glycogen and its metabolism. Curr Mol Med 2002; 2: 101–20. 19. Giatromanolaki A. Prognostic role of angiogenesis in non-small cell lung cancer. Anticancer Res 2001; 21: 4373–82. 20. Matsuyama K, Chiba Y, Sasaki M, Tanaka H, Muraoka R, Tanigawa N. Tumor angiogenesis as a prognostic marker in operable non-small cell lung cancer. Ann Thorac Surg 1998; 65: 1405–9. 21. Meert AP, Paesmans M, Martin B, et al. The role of microvessel density on the survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer 2002; 87: 694–701. 22. Offersen BV, Pfeiffer P, Hamilton-Dutoit S, Overgaard J. Patterns of angiogenesis in non small-cell lung carcinoma. Cancer 2001; 91: 1500–9. 23. Tsoli E, Zacharatos P, Dasiou-Plakida D, et al. Growth index is independent of microvessel density in non-small-cell lung carcinomas. Hum Pathol 2002; 33: 812–8. 24. Hu J, Bianchi F, Ferguson M, et al. Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer. Oncogene 2005; 24: 1212–9. 25. Oyama T, Osaki T, Nose N, et al. Evaluations of p53 immunoreactivity, nucleolar organizer regions, and proliferating cell nuclear antigen in non-small cell lung carcinoma. Anticancer Res 2000; 20: 505–10. 26. Poleri C, Morero JL, Nieva B, et al. Risk of recurrence in patients with surgically resected stage I non-small cell lung carcinoma: histopathologic and immunohistochemical analysis. Chest 2003; 123: 1858–67. 27. Nguyen VN, Mirejovsky P, Mirejovsky T, Melinova L, Mandys V. Expression of cyclin D1, Ki-67 and PCNA in non-small cell lung cancer: prognostic significance and comparison with p53 and bcl-2. Acta Histochem 2000; 102: 323–38. 28. Grossi F, Loprevite M, Chiaramondia M, et al. Prognostic significance of K-ras, p53, bcl-2, PCNA, CD34 in radically resected non-small cell lung cancers. Eur J Cancer 2003; 39: 1242–50. 29. Mathupala SP, Rempel A, Pedersen PL. Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase. J Bioenerg Biomembr 1997; 4: 339–43. 30. Schnier JB, Nishi K, Monks A, Gorin FA, Bradbury EM. Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression. Biochem Biophys Res Commun 2003; 309: 126–34.
ANATOMICAL PATHOLOGY
Clinicopathological significance of BGP expression in non-small-cell lung carcinoma: relationship with histological type, microvessel density and patients’ survival MIN KI LEE*{, JAE HO KIM{, CHANG HUN LEE*§, JONG MIN KIM**, CHI DUK KANGd, YOUNG DAE KIM", KYUNG UN CHOI§, HWAL WOONG KIM§, JEE YEON KIM§, DO YOUN PARK§ AND MEE YOUNG SOL§ *Medical Research Institute, and Departments of {Internal Medicine, {Physiology, §Pathology, dBiochemistry and "Thoracic Surgery, College of Medicine, Pusan National University, Busan, Korea; **Department of Anatomy, College of Medicine, Dong A University, Busan, Korea
Summary Aims: Brain-type glycogen phosphorylase (BGP) is the major isoform of glycogen phosphorylase found in fetal and neoplastic tissues, and is generally thought to induce glucose supply during an ischaemic period. This study was performed to investigate BGP expression in non-small-cell lung carcinoma (NSCLC). Methods: A total of 119 cases of NSCLC, including 63 squamous cell carcinomas (SqCCs) and 56 adenocarcinomas (ACs), were imunohistochemically evaluated for BGP expression, and its expression was correlated with clinicopathological parameters. Results: In total, 76.5% were positive, while non-neoplastic bronchial epithelial cells were weakly positive and pneumocytes were negative. High BGP expression was noted in 78.6% of ACs and 36.5% of SqCCs (p50.001). Microvessel density was higher in the low BGP expression tumours (29.6 ¡ 16.9/mm2) than in the high expression tumours (22.8 ¡ 13.8/mm2) (p50.017). BGP expression did not correlate with patient age or tumour stage, but was more frequent in females than males. Kaplan–Meier analysis showed that high BGP expression was associated with poorer survival (p50.032). Conclusions: BGP is expressed in NSCLC, particularly AC, and is an independent poor prognostic factor. Key words: Brain-type glycogen phosphorylase, BGP, non-small-cell lung carcinoma, NSCLC, histology, microvessel density, survival. Received 4 June, revised 18 August, 5 September, accepted 11 September 2006
INTRODUCTION In epithelial cells of the developing respiratory tract, mobilisation of glycogen stores is regulated differentially such that glycogenolysis in the alveolar epithelium generally precedes that in the bronchial and bronchiolar epithelium.1 Glycogen phosphorylase (GP) catalyses the phosphorolysis of glycogen to glucose 1-phosphate, which represents the initial step for the synthesis of pulmonary surfactant phospholipids in alveolar epithelium of the developing airway. Mammalian GPs are found in three major isozymes, i.e., muscle, liver and
brain, that can be distinguished by functional and structural properties as well as by the tissues in which they are predominantly expressed.2–4 cDNA sequences encoding the three human GP isoforms have been aligned.3,5 Although they have very similar enzymatic activities and primary structures (80–90% homology at amino acid level), chromosomal mapping analyses have revealed that the genes encoding muscle, liver, and brain-type GP (BGP) are assigned to chromosomes 11, 14, and 20, respectively.3 The physiological role of BGP is generally thought to be the provision of an emergency glucose supply during a stressful and ischaemic period, as may occur in neoplasms. Of note, the major isoform of GP found in fetal and tumour tissues is BGP.6,7 Most reported studies of BGP expression in human cancers focus on gastrointestinal tract carcinoma.8–10 In colorectal carcinoma, the immunohistochemical expression of BGP has been reported to be common, with significant increase during the colorectal adenoma-carcinoma sequence. In particular, positive BGP staining in noncancerous colorectal mucosa (called BGP foci) was observed mainly around adenocarcinomas without an adenomatous component. These results led to the suggestion that the BGP focus might contribute to carcinogenesis.8 In gastric cancer, a strong correlation was observed between BGP expression and both intestinaltype carcinoma and intestinal metaplasia, whereas no positive staining was noted in the normal gastric epithelial cells.9,11 Compared with these gastrointestinal carcinomas, BGP expression has not previously been reported in nonsmall-lung carcinoma (NSCLC). This study is designed to investigate for the first time the immunohistochemical localisation of BGP in NSCLC, and then evaluate the relationship between BGP expression and clinicopathological factors of NSCLC.
MATERIALS AND METHODS Patients and clinicopathological data All patients with NSCLC who had undergone lobectomy or pneumonectomy at Pusan National University Hospital (PNUH), Korea, during the
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020601024029
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period January 1998 to December 2002 were considered. After exclusion of cases in which there was insufficient pathological material remaining for further study, a total of 119 cases were selected for this study. There were 96 men and 23 women in the study population. Mean age was 58 years (range 27–79 years). Tumours were staged according to the TNM classification of the International Union Against Cancer (UICC) staging system12 after reviewing the clinical, radiological, and pathological data. Other clinical information was extracted from medical records. For survival analysis, the follow-up information was also checked. Patient survival was calculated as the time between operation and death. Patients who were still alive at the time of data collection were censored in the statistical analysis. The surgically resected specimens were fixed immediately in 10% buffered formalin (pH 7.0). All sections containing both tumour tissue and surrounding lung tissue were embedded in paraffin, and five serial sections (4 mm) were cut from a selected, paraffin-embedded tissue block. For pathological diagnosis, one of these sections was stained with H&E. The others were used for immunohistochemistry. Pathological diagnosis was based on the third edition of the World Health Organization classification.13 The tumours included 63 cases of squamous cell carcinoma (SqCC) and 56 of adenocarcinoma (AC). Additionally, part of the fresh specimens from cancer foci and their surrounding lung tissues were frozen at 280uC for Western blot analysis. Immunohistochemistry Sections from paraffin-embedded blocks were transferred to poly-L-lysine coated glass slides and air-dried overnight at 37uC. They were dewaxed in xylene (three changes), rehydrated in a graded series of decreasing ethanol concentrations, and then rinsed in Tris-buffered saline (50 mM Tris/HCl, pH 7.4, containing 100 mM sodium chloride). Endogenous peroxidase activity was inactivated with 5% hydrogen peroxide in methanol for 15 min at 37uC. The following primary antibodies were used: anti-BGP (rabbit polyclonal anti-human BGP, 1:500), anti-CD34 (mouse monoclonal antiCD34, QBEnd/10, 1:50; NeoMarkers, USA), and anti-proliferating cell nuclear antigen (PCNA; mouse monoclonal anti-PCNA, PC10, 1:1 500; Sigma Chemical Co., USA). Anti-BGP antibody was a generous gift from Dr Michio Ogawa (Department of Surgery II, Kumamoto University School of Medicine, Japan). Each primary antibody was incubated with the tissue sections overnight at 4uC, and then immunohistochemical procedures were performed using the Histostain Plus kit (Zymed Laboratories, USA) using the standard streptavidin-biotin complex method. The reaction products were visualised by exposing sections to 3-amino9-ethylcarbazole. Nuclei were lightly counterstained for about 20 s with Mayer’s haematoxylin. Sections were then mounted in diluted malinol after the application of Universal Mount (Dako, USA). In each staining batch, appropriate positive control specimens were used. Tissue samples incubated with non-immune serum served as negative controls. Evaluation of immunohistochemical stainings All immunohistochemical evaluation was performed by two independent observers (CHL and MKL). Discordance between the observers was resolved by consensus using a multiheaded microscope. For BGP, immunostaining was semiquantitatively graded on a scale of 0 to 3+ according to area percentage of cells with positive cytoplasmic staining: 0 for absent or less than 10%; 1+ for 10–25%; 2+ for 26–50%; and 3+ for more than 50%. Grades 0 and 1 were regarded as low expression, and grades 2 and 3 as high expression. CD34 immunostaining was performed to evaluate tumour angiogenic activity expressed as microvessel density (MVD), and CD34-positive microvessels were assessed in the surrounding stroma of invasive tumour nests as described in the literature.14 Briefly, in four adjacent fields of vision in the most vascularised area, microvessels were counted at 6200 magnification using an Olympus microscope (BX 50; Olympus, Japan), and then MVD was expressed as the mean value of microvessels/mm2 for each case. For PCNA, we examined approximately 1000 tumour cells from each tumour tissue for nuclear positivity at high power (6400) after screening for areas of highest staining intensity at low power (6100). The labelling index for PCNA was
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expressed as the percentage of cells showing positive nuclear staining in each tumour. Western blot analysis for BGP expression in normal lung and tumour tissues NSCLC tumour tissues (10 cases of AC and 10 cases of SqCC) and their respective normal lung tissues were homogenised for 3 min on ice in lysis buffer (20 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, 10 mM NaCl, 0.1 mM PMSF, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 25 mMglycerol phosphate, 1% Triton X-100, pH 7.4). The homogenates were centrifuged at 10 000 g for 10 min, and the supernatants were used as the protein extracts of tissues. The protein concentrations of the extracts were determined using the Bio-Rad protein assay kit (Bio-Rad, USA). On 10% SDS-polyacrylamide gel, 60 mg of the extract was resolved and electrotransferred to nitrocellulose membrane. The membrane was blocked in 5% non-fat milk in TBST (20 mM TrisHCl, 137 mM NaCl, 0.1% Tween 20, pH 7.6) and incubated with anti-BGP antibody (rabbit polyclonal anti-BGP, 1:2000) for 2 h at room temperature. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000; Jackson ImmunoResearch Laboratories, USA) for 1 h at room temperature. Immunoreactive proteins were detected with the enhanced chemiluminescence detection system (Amersham Biosciences, UK). Statistical analysis The Pearson x2 and Student’s t tests were used to evaluate whether BGP expression correlated with clinicopathological parameters. The Mann– Whitney U test was also used for quantitative comparison of BGP expression between histological types of NSCLC after densitometric conversion of Western blot bands. For univariate survival analyses, the Kaplan–Meier method was applied, and the difference between the survival curves was analysed by the log-rank test. The Cox proportional hazard model was used to estimate the hazard ratio (HR) of independent factors influencing patient’s survival. All analyses were performed on a personal computer with the SPSS statistical package (Release 12.0.1; SPSS Inc., USA). p values less than 0.05 were regarded as statistically significant. All statistical tests were twosided.
RESULTS BGP expression in non-neoplastic lung and carcinoma tissues Bronchial epithelial cells, bronchial glands and bronchial smooth muscle showed weak positive immunoreactivity to anti-BGP antibody (Fig. 1A). Alveolar epithelial cells (pneumocytes), stromal fibroblasts and inflammatory cells were negative. Immunoreactivity to anti-BGP antibody was found in 76.5% (91/119) of NSCLC cases. In AC, carcinoma cells usually showed diffuse strong positivity (Fig. 1B). Nonneoplastic alveolar epithelial cells were negative, which contrasted with adjacent carcinoma showing distinctive positive reaction (Fig. 1C). In SqCC, immunoreactivity was generally more heterogeneous in distribution and intensity (Fig. 1D). When the cancers were divided into two groups according to the levels of BGP expression, 43.7% (52/119) of cancers showed low BGP expression and 56.3% (67/119) showed high expression. Relationship between BGP expression and clinicopathological parameters As shown in Table 1, histological type, MVD, and patient sex correlated with BGP expression. High expression of BGP was present in 78.6% (44/56) of AC, in contrast to 36.5% (23/63) of SqCC. The level of BGP expression was
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Fig. 1 (A) In non-neoplastic lung tissue, BGP expression is weakly positive in bronchial epithelial cells, but negative in alveolar epithelial cells (6100). (B) Adenocarcinoma showing diffuse BGP expression (6200). (C) Area of adenocarcinoma with strong BGP expression is sharply contrasted with non-neoplastic alveolar cells showing negative BGP reaction (6200). (D) Squamous cell carcinoma showing focal BGP expression (6200). (E,F) CD34-postive microvessels (E) in adenocarcinoma with low BGP expression and (F) in squamous cell carcinoma with high BGP expression (6200). (G,H) PCNA expression (G) in adenocarcinoma with low BGP expression and (H) in squamous cell carcinoma with high BGP expression (6200).
significantly higher in ACs than in SqCCs (p,0.001) using the x2 test. The degree of differentiation in both histological types was not associated with the level of BGP expression. MVD, assessed by CD34 immunoreactivity, revealed a significant difference between the low and high expression groups of BGP (29.6¡16.9 microvessels/mm2 in the former versus 22.8¡13.8 microvessels/mm2 in the latter, p50.017; Fig. 1E,F; Table 1) using the Student’s t test. The PCNA index of the low BGP expression group was slightly higher
than that of high BGP group, although this was not significant (p50.408) using the x2 test (Fig. 1G,H). Regarding patient sex, high expression of BGP was present in 78.3% (18/23) of tumours in female patients and in 51.0% (49/96) of male patients. The BGP expression of female patients was significantly higher than that of male patients using the x2 test (p50.018). Otherwise, the level of BGP expression in NSCLC did not correlate with patient age or stage using the x2 test (p.0.05, respectively; Table 1).
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TABLE 1 Relationship between BGP expression and clinicopathological parameters in non-small-cell lung carcinomas BGP expression
Parameters Histological type SqCC AC Age (years) (58* .58 Sex Female Male Stage I II III TNM-pT T1 T2 T3-4 TNM-pN N0 N1-2 MVD (/mm2){ PCNA (%){
Total no. cases
Low (n552)
High (n567)
63 56
40 12
23 44
54 65
23 29
31 36
23 96
5 47
18 49
56 19 44
27 11 14
29 8 30
23 68 28
11 32 9
12 36 19
68 51 119 119
32 20 29.6¡16.9 49.9¡21.3
36 31 22.8¡13.8 46.5¡22.6
p value ,0.001 0.825 0.018 0.103
0.489
0.393 0.017 0.408
*Mean age group to create groups. {Expressed as mean¡SD. SqCC, squamous cell carcinoma; AC, adenocarcinoma; MVD, microvessel density; PCNA, anti-proliferating cell nuclear antigen.
Quantitative comparison of BGP expression according to histological types In Western blot analyses using the extracts from 20 NSCLCs (10 cases of AC and 10 of SqCC) and corresponding normal lung tissues, anti-BGP antibody showed the formation of characteristic 107 kDa bands, consistent with BGP molecule, in all materials (Fig. 2). When densitometry (LabWorks Software 4.0; Ultra-Violet Products, UK) was applied for the calculation of relative density ratios for tumour to normal bands, the ratios of ACs were significantly higher than those of SqCCs using the Mann–Whitney U test (p50.001; Fig. 3).
Fig. 3 Box plots of the values of tumour-to-normal relative ratios after densitometric calculation of BGP-specific bands from Western blot analyses. SqCC, squamous cell carcinoma; AC, adenocarcinoma; NSCLC, non-small-cell lung carcinoma.
survival was defined as the interval in months between the day of surgical resection and date of either death or the last follow-up. An observation was censored at the last followup when the patients were alive or had died from a cause other than cancer. With regard to BGP expression, 26 of 47 patients with low expression were censored and 21 died (mean and median survival times, 66.9 and 77.0 months, respectively). Twenty of 63 patients with high expression were censored and 43 died (mean and median survival times, 44.9 and 25.0 months, respectively). When Kaplan–Meier’s univariate analysis was performed, the overall survival was associated with the level of BGP expression (p50.032 by log-rank test; Fig. 4), but not with histological type, MVD, and patient sex (p50.581, 0.180, and 0.603, respectively). A Cox proportional hazard regression model also identified BGP expression as a single independent prognostic factor (hazard ratio 1.748; 95%
Survival analysis During the course of this study (maximal follow-up 132 months; minimal follow-up 1 month; median follow-up 23 months), nine cases were lost to follow-up. The length of
Fig. 2 Western blot analysis using rabbit polyclonal anti-human antibody to BGP in non-small-cell lung carcinomas. A single band is observed at 107 kDa in all samples of normal and tumour tissues, although its density is rather varied to the types of samples. Lanes 1–5, adenocarcinoma; lanes 6– 10, squamous cell carcinoma; N, non-neoplastic lung tissue; T, tumour tissue.
Fig. 4 Overall survival curves of patients with non-small-cell lung carcinoma showing low expression (n547) and high expression (n563) of BGP.
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confidence interval 1.037–2.948; p50.036), after controlling for potential confounding factors such as histological type, MVD, sex, age, and PCNA index.
DISCUSSION Glycogen accumulates to significant levels in epithelial cells of the developing respiratory tract. Glucose, originating from glycogen, provides substrate for the synthesis of fatty acid and glycerol which, in turn, are incorporated into surfactant dipalmitoyl phosphatidylcholine.15 Rannels et al.16 suggested that BGP activity was clearly detected in isolated fetal type II pneumocytes of developing rat lung and might be involved in mobilisation of type II cell glycogen during late fetal lung development. Glucose uptake and metabolism are essential for proliferation and survival of cells, and are thought to be enhanced in actively proliferating cell systems such as developing fetal lung epithelium and lung carcinomas.17,18 Thus, glycogenolysis by BGP may have an important role in tumour cell growth. However, the clinicopathological significance of the glycolytic enzyme BGP in NSCLC has not been described so far in the English literature. In the present study, positive immunoreactivity to antiBGP antibody was found in 76.5% (91/119) of NSCLC cases and the level of BGP expression was significantly higher in ACs than in SqCCs (p,0.001). Western blot data also showed that lung AC tissues contained distinctively enhanced tissue levels of BGP, whereas SqCC tissues usually had slightly higher levels of BGP compared with those of the corresponding non-neoplastic lung tissues. In normal lung tissues, pneumocytes were negative for antiBGP antibody. Normal bronchiolar and bronchial cells frequently showed diffuse positivity for the antibody, although the intensity of staining was usually weaker than in carcinoma cells. Tashima et al.8 suggested that the expression of BGP in colorectal cancers may be indicative of oncofetal antigen reversion in cancer, like a-fetoprotein in hepatocellular carcinomas or carcinoembryonic antigen in gastrointestinal cancers. They found that BGP was expressed in the colonic adenomas with higher grades of dysplasia and cancers, and there was positive correlation between BGP expression and the adenoma-carcinoma sequence. We could not evaluate BGP expression in preneoplastic stages of AC, but we did find that the degree of histological differentiation had no relationship to BGP expression. Our immunohistochemical and Western blot expression data might be explained by oncofetal conversion as postulated in gastrointestinal carcinomas. The cellular expression of BGP in SqCC could be associated partly with oncogenic over-expression of the molecule, although its exact biological mechanism is still not determined. Previous studies of the prognostic role of angiogenesis in NSCLC have been contradictory, with some workers finding poorer outcomes in tumours with high levels of angiogenesis19–21 and others finding no correlation.22–24 In our study we could not find any association between MVD and overall survival of NSCLC patients. However, we did find that the MVD of the low BGP expression group was significantly higher than that of the high BGP expression group, suggesting that BGP expression is increased
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as a result of tumour hypoxia. It appears that angiogenesis and BGP expression are independent variables in NSCLC. In the present study with NSCLCs, the overall survival time of the high BGP expression group was significantly shorter than that of the low BGP expression group. A multivariate analysis also confirmed that the level of BGP expression was an independent prognostic factor. There was no relationship between stage and the level of BGP expression. There was significantly higher BGP expression in the tumours of female patients than in males. Given that BGP expression is higher in AC than in SqCC, the sex difference is most likely explained on the basis of the higher incidence of AC in females. Uncontrolled cell proliferation is the hallmark of malignant tumours. However, the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial.25–28 The role of glycogen metabolism in cell proliferation and tumour formation is not well studied, even though altered regulation of carbohydrate metabolism has long been associated with cancer cells.29 Schnier et al.30 showed that when A549 NSCLC cells expressing BGP were treated by the GP inhibitor CP-91,149, the cells accumulated glycogen and demonstrated growth inhibition. Unexpectedly, the present study found that the PCNA index of tumours from the low BGP expression group was slightly higher than that of the high BGP group, although the result did not reach significance. This suggests that the requirement for glucose to provide energy for cell growth is not the sole reason for BGP expression. Instead, BGP expression in NSCLC might represent oncofetal conversion or over-expression phenomenon during carcinogenesis. In summary, we have described the incidence of BGP expression in non-small-cell lung carcinoma and demonstrated its significant association with adenocarcinoma and poor survival outcome. Further study is necessary to elucidate the underlying pathobiology of this association and the role, if any, of BGP in lung carcinogenesis. ACKNOWLEDGEMENTS The authors thank Dr Michio Ogawa (Department of Surgery II, Kumamoto University School of Medicine, Japan) for kindly providing rabbit polyclonal anti-human BGP antibody in the present study. We also thank Young Jae Lee for excellent technical assistance. This study was supported by Medical Research Institute Grant (2004-40), Pusan National University, Busan, Korea. Address for correspondence: Dr C. H. Lee, Department of Pathology, College of Medicine, Pusan National University, 1-10 Ami-dong, Seo-gu, Busan 602-739, Korea. E-mail: [email protected]
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