AJCP / Original Article
Expression of Metabolism-Related Proteins in Lacrimal Gland Adenoid Cystic Carcinoma Ja Seung Koo, MD, PhD,1 and Jin Sook Yoon, MD, PhD2 From the 1Department of Pathology and 2Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, South Korea. Key Words: Adenoid cystic carcinoma; Lacrimal gland; Metabolism-related protein; Protein expression; Salivary gland Am J Clin Pathol April 2015;143:584-592 DOI: 10.1309/AJCPXAYH10WENLTC
ABSTRACT Objectives: To investigate the expression and the clinical implications of metabolism-related proteins in lacrimal gland adenoid cystic carcinoma (ACC) in comparison with salivary gland ACC. Methods: Human tissue samples of lacrimal gland ACC (n = 11) and salivary gland ACC (n = 64) were analyzed. Immunochemistry was used to measure expression of proteins related to glycolysis (glucose transporter 1, hexokinase II, carbonic anhydrase IX, and monocarboxylate transporter 4 [MCT4]), glutaminolysis (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter 2 [ASCT2]), mitochondria (adenosine triphosphate [ATP] synthase, succinate dehydrogenase A [SDHA], and succinate dehydrogenase B), and glycolytic intermediate metabolism (phosphoserine phosphatase [PSPH], serine hydroxymethyl transferase 1 [SHMT1]). Results: GLS1 and ASCT2 were more highly expressed, and GDH, ATP synthase, and SDHA were expressed to a lesser degree in lacrimal gland ACC than in salivary gland ACC (P < .05). Lacrimal gland ACC showed less of a mitochondrial phenotype than did salivary gland ACC (P = .001). Positivity of MCT4 and PSPH was related to shorter disease-free survival, and SHMT1 was related to shorter overall survival (P < .05). Conclusions: Lacrimal gland ACC exhibited higher expression of GLS1 and ASCT2, compared with salivary gland ACC. Overexpression of MCT4, PSPH, and SHMT1 was associated with poorer prognosis.
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Adenoid cystic carcinoma (ACC) is a malignancy of secretory glands, including the lacrimal and salivary glands, with a poor prognosis and difficult decisions for treatment.1 Despite a slow rate of growth, lacrimal gland ACCs are highly aggressive and prone to local recurrence and perineural spread to the cranial nerves.1 ACC represents the most common malignancy of the lacrimal glands, comprising 3.8% of primary ocular adnexal tumors.2 ACC accounts for approximately 40% of salivary gland malignancies and is characterized by a slow but persistent progression, with multiple local recurrence and metastasis to the lung, bone, and brain occurring in approximately 50% of patients.3,4 There are only a few studies of lacrimal gland ACC, in comparison with salivary gland ACC, due to rarity of the tumor. Factors related to poor prognosis of salivary gland ACC include advanced stage, solid architecture, high histologic grade, perineural invasion, and positive surgical resection margin.5-7 Compared with salivary gland ACC, lacrimal gland ACC has shown clinical features of younger age and poorer prognosis.8 The causes of poorer prognosis of lacrimal gland ACC include the high incomplete excision rate due to complex orbital anatomy, invasion to nearby structures, and subsequent metastases.1,9 Primary treatments of lacrimal gland ACC are en bloc surgical excision and postoperative radiation, as needed.10 Tse et al11 recently reported that neoadjuvant intra-arterial cytoreductive chemotherapy improved overall survival and decreased recurrence in 19 lacrimal gland ACCs. However, controversy regarding treatment still exists.12 Neutron radiation therapy achieved 80% of 5-year local control in 11 cases, although late recurrence and distant metastasis remained as challenges.13 There is no effective treatment
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AJCP / Original Article
❚Table 1❚ Clone Source, Dilutions Used, Sources of Antibodies Used, and Expression Pattern Antibody Glycolysis related Glut1 Hexokinase II CAIX MCT4 Glutaminolysis related GLS1 GDH ASCT2 Mitochondrial related ATP synthase SDHA SDHB Serine/glycine metabolism related PHGDH PSAT1 PSPH SHMT1 GLDC
Clone
Dilution Source
Expression Pattern
SPM498 Polyclonal Polyclonal Polyclonal
1:200 1: 100 1:100 1:100
Abcam, Cambridge, England Abcam Abcam Santa Cruz Biotech, Santa Cruz, CA
Cytoplasmic and/or membranous Cytoplasmic Cytoplasmic Membranous
Polyclonal Polyclonal Polyclonal
1:50 1:100 1:100
Abcam Abcam Abcam
Cytoplasmic Cytoplasmic Membranous
15H4C4 1:100 2E3GC12FB2AE2 1:100 21A11AE7 1:100
Abcam Abcam Abcam
Cytoplasmic Cytoplasmic Cytoplasmic
Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal
Abcam Abcam Abcam Abcam Abcam
Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic
1:100 1:100 1:100 1:100 1:100
ASCT2, amino acid transporter 2; ATP, adenosine triphosphate; CAIX, carbonic anhydrase IX; GDH, glutamate dehydrogenase; GLDC, glycine dehydrogenase, decarboxylating; GLS1, glutaminase 1; Glut1, glucose transporter 1; MCT4, monocarboxylate transporter 4; PHGDH, phosphoglycerate dehydrogenase; PSAT1, phosphohydroxythreonine aminotransferase 1; PSPH, phosphoserine phosphatase; SDHA, succinate dehydrogenase A; SDHB, succinate dehydrogenase B; SHMT1, serine hydroxy methyltransferase 1.
modality in cases of recurrence or metastasis of lacrimal gland ACC. New treatments targeting this rare and lifethreatening cancer are needed. One of the hallmarks of cancer is metabolic deregulation, which has been recently discussed and investigated as a possible therapeutic target to treat cancer.14 Many tumors display increased glucose uptake and metabolism through the process of aerobic glycolysis, also known as the Warburg effect, suggesting that glycolysis is a major component of tumor metabolism.15 However, together with glycolysis, cancer cells use several metabolic systems, including glutaminolysis, mitochondria metabolism, and glycolytic intermediate metabolism.16,17 Thus far, no studies have investigated the characteristics of metabolism in lacrimal gland ACC. The purpose of this study was to demonstrate the expression of proteins related to several metabolic pathways, including glycolysis, glutaminolysis, mitochondrial metabolism, and glycolytic intermediate metabolism in lacrimal gland ACC in comparison with salivary gland ACC, and to find prognostic factors associated with survival.
Materials and Methods Patient Selection and Clinicopathologic Evaluation Formalin-fixed, paraffin-embedded tissue samples of lacrimal gland ACC, collected from January 1997 through December 2012, at Severance Hospital in Seoul, South Korea, were used for analyses. The study was approved by
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the Institutional Review Board of Severance Hospital and adhered to the tenets of the Declaration of Helsinki. Clinical information such as age at surgery, sex, tumor side, symptoms, and visual acuity were informed from medical chart recordings. Tumor stage classifications followed the seventh American Joint Committee on Cancer staging system, and histologic features of H&E slides were reviewed by a specialized pathologist (J.S.K.). Histologic grading of tumors followed the indications established by Szanto et al18 as follows: grade I, no solid component; grade II, ACC with less than 30% solid component; and grade III, ACC with more than 30% solid component. Histologic type was determined by predominant morphologic growth patterns and divided into cribriform, tubular, and solid patterns. Perineural invasion, tumor margin (expanding, infiltrative), and tumor involvement in the surgical resection margin were evaluated. As a control group, 64 cases of major salivary gland ACC were included in the same periods of tissue collection. Immunohistochemistry The antibodies used for immunohistochemistry (IHC) in this study are listed in ❚Table 1❚. IHC was performed using the Ventana Discovery XT automated staining system (Ventana Medical Systems, Tucson, AZ) following the manufacturer’s protocols. Briefly, Slides of 3-µm–thick tissue sections were incubated for 1 hour at room temperature in standard Cell Conditioning 1 buffer (citrate buffer, pH 6.0; Ventana Medical Systems). The tissue samples were subsequently incubated with primary antibodies for 32 minutes at room temperature, washed, and incubated with the
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Koo et al / Protein Expression in Lacrimal Gland ACC
❚Table 2❚ Clinicopathologic Characteristics of Lacrimal Gland Adenoid Cystic Carcinoma Age, Case No. y/Sex
Tumor Size, Main Histologic Histologic cm/Tumor Side Type Grade
Lymphovascular Ocular Invasion Symptoms
Local Chemotherapy/ Recurrence/ Radiation Metastasis Survival Therapy
1 2 3 4 5 6 7 8 9 10 11
3.0/R 2.6/L 4.0/L 3.5/R 3.5/R 3.0/R 2.5/L 3.6/R 3.0/R 3.5/L 2.5/R
No No No No No No Yes No Yes No No
Yes/yes No/yes Yes/yes No/yes No/no Yes/no No/no No/yes No/no Yes/yes Yes/yes
41/F 21/M 63/F 54/M 28/M 72/M 57/M 51/M 35/M 61/F 43/M
Cribriform Cribriform Solid Solid Tubular Solid Cribriform Tubular Cribriform Tubular Cribriform
2 1 3 3 1 3 1 2 1 2 1
ultraView Universal DAB kit (Ventana Medical Systems) for 16 minutes at room temperature. Then, tissue sections were stained with Hematoxylin ReaDi solution (Ventana Medical Systems). IHC included the appropriate positive and negative controls. IHC was performed to measure expression of proteins related to glycolysis (glucose transporter 1 [Glut1], hexokinase II, carbonic anhydrase IX [CAIX], and monocarboxylate transporter 4 [MCT4]), glutaminolysis (glutaminase 1 [GLS1] and glutamate dehydrogenase [GDH]), amino acid transporter 2 (ASCT2), mitochondrial metabolism (adenosine triphosphate [ATP] synthase, succinate dehydrogenase A [SDHA], and succinate dehydrogenase B [SDHB]), and glycolytic intermediate metabolism (phosphoglycerate dehydrogenase [PHGDH], phosphohydroxythreonine aminotransferase 1 [PSAT1], phosphoserine phosphatase [PSPH], serine hydroxymethyl transferase 1 [SHMT1], and glycine dehydrogenase decarboxylase [GLDC]) in 11 cases of lacrimal gland ACC and 64 cases of major salivary gland ACC. Interpretation of Immunohistochemical Results Results of IHC were defined as the proportion of stained cells × immunostaining intensity. The proportion of stained cell was defined as follows: 0, negative; 1, less than 30% positivity; and 2, 30% or more positivity. Immunostaining intensity was defined as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The proportion of stained cells × immunostaining intensity was defined as follows: 0 to 1 was negative, and 2 to 6 was positive.19 Classification of Tumor Metabolic Subtype Tumor metabolic subtype was classified according to the expression pattern of metabolism-related proteins: (1) glycolysis type (more than three positive expressions in Glut1, hexokinase II, CAIX, and MCT4) and nonglycolysis type, (2) glutaminolysis type (more than two positive expressions in GLS1, GDH, and ASCT2) and nonglutaminolysis
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Proptosis Proptosis Lid swelling Proptosis Proptosis Proptosis Palpable mass Proptosis Proptosis Proptosis Lid swelling
Dead Alive Dead Dead Alive Alive Alive Alive Alive Alive Alive
No/yes No/yes No/no No/yes Yes/yes Yes/yes No/yes Yes/yes Yes/yes Yes/yes Yes/yes
type, (3) mitochondrial type (more than two positive expressions in ATP synthase, SDHA, and SDHB) and nonmitochondrial type, and (4) serine/glycine type (more than three positive expressions in PHGDH, PSAT1, PSPH, SHMT1, and GLDC) and nonserine/glycine type. Statistical Analysis Data were statistically processed using SPSS for Windows version 12.0 (SPSS, Chicago, IL). The Student t test and Fisher exact test were used for continuous and categorical variables, respectively. To analyze data with multiple comparisons, a corrected P value with application of the Bonferroni method for multiple comparisons was used. Statistical significance was defined as P < .05. Kaplan-Meier survival curves and log-rank statistics were employed to evaluate survival time and time to tumor metastasis, respectively. Multivariate regression analysis was performed using the Cox proportional hazards model.
Results Basal Characteristics of Lacrimal Gland ACC Initially, we found that 14 patients received an operation for lacrimal gland ACC in our database, but since the histologic analysis of the tissue block from three patients was impossible, this study included 11 cases of lacrimal gland ACC ❚Table 2❚. Clinical characteristics included age from 21 to 72 years, with three women and eight men. The size of the tumor was 2.5 to 4.0 cm, and the main histologic type was cribriform (n = 5). The most common histologic grade was grade 1 (n = 5). Perineural invasion was not found in all cases. Lymphovascular invasion was observed in two cases, local recurrence in five cases, and distant metastases in seven cases (brain, n = 5; lung, n = 2). Three patients died of disease.
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AJCP / Original Article
❚Table 3❚ Comparisons of the Expression of Metabolism-Related Proteins Between Lacrimal Gland and Salivary Gland Adenoid Cystic Carcinoma (ACC)a Parameter
Total (n = 75)
Lacrimal Salivary Gland ACC Gland ACC (n = 11) (n = 64)
P Value
Glut-1 (T) Hexokinase II (T) CAIX (T) MCT4 (T) MCT4 (S) GLS1 (T) GDH (T) ASCT2 (T) ATP synthase (T) SDHA (T) SDHB (T) PHGDH (T) PSAT1 (T) PSPH (T) SHMT1 (T) SHMT1 (S) GLDC (T)
27 (36.0) 7 (9.3) 17 (22.7) 14 (18.7) 9 (12.0) 15 (20.0) 64 (85.3) 6 (8.0) 63 (84.0) 60 (80.0) 39 (52.0) 66 (88.0) 9 (12.0) 4 (5.3) 65 (86.7) 26 (34.7) 7 (9.3)
6 (54.5) 2 (18.2) 3 (27.3) 3 (27.3) 1 (9.1) 5 (45.5) 4 (36.4) 3 (27.3) 5 (45.5) 4 (36.4) 4 (36.4) 11 (100.0) 0 (0.0) 2 (18.2) 8 (72.7) 6 (54.5) 0 (0.0)
.188 .272 .704 .420 1.000 .037 3 cm
❚Figure 1❚ Correlations between metabolism-related proteins and clinicopathologic factors in lacrimal gland adenoid cystic carcinoma. All cases with tumor sizes 3 cm or less (n = 6) were adenosine triphosphate (ATP) synthase negative, whereas all cases with tumor size more than 3 cm (n = 5) expressed more ATP synthase.
addition, this study could not demonstrate many significant findings despite numerous analyses. Additional studies are warranted to confirm the different expression of these proteins between lacrimal gland ACC and salivary gland ACC.
Conclusions We have demonstrated for the first time that lacrimal gland ACC showed distinct profiles of several metabolismrelated protein expression. Compared with salivary gland ACC, lacrimal gland ACC exhibited lower expression of GDH, ATP synthase, and SDHA and higher expression of ASCT2 and GLS1. Metabolic regulatory proteins have emerged as attractive targets for cancer therapy. For example, clinical and preclinical studies of inhibitors of Glut1,33 PHGDH,34 and GDH35 have been evaluated. Our data may provide further insights and a basis for further studies to identify potential cancer targets from among the metabolism-related proteins in lacrimal gland ACC. Because lacrimal gland ACC is fatal, has a poor prognosis, and is without available and effective chemotherapeutic agents, identification of novel therapeutic drugs targeting these metabolic regulators in lacrimal gland ACC is essential. Address reprint requests to Dr Yoon: Dept of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Severance Hospital, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, South Korea; email: [email protected]. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2012R1A1A1002886). The funding organization had no role in the design or conduct of this research.
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❚Table 5❚ Univariate Analyses by Log-Rank Tests of the Impact of Metabolism-Related Proteins on Disease-Free Survival and Overall Survival in Lacrimal Gland Adenoid Cystic Carcinomaa Disease-Free Survival Parameter Glut-1 (T) Negative Positive Hexokinase II (T) Negative Positive CAIX (T) Negative Positive MCT4 (T) Negative Positive MCT4 (S) Negative Positive GLS1 (T) Negative Positive GDH (T) Negative Positive ASCT2 (T) Negative Positive ATP synthase (T) Negative Positive SDHA (T) Negative Positive SDHB (T) Negative Positive PHGDH (T) Negative Positive PSAT1 (T) Negative Positive PSPH (T) Negative Positive SHMT1 (T) Negative Positive SHMT1 (S) Negative Positive GLDC (T) Negative Positive
No. of Months (95% CI)
P Value
Overall Survival No. of Months (95% CI)
.929 41 (24-59) 36 (18-53)
P Value .180
68 (54-81) 245 (245-245) .977
41 (24-58) 44 (26-61)
.379 80 (73-86) 141 (0-344)
.390 46 (31-62) 28 (0-57)
.431 74 (61-86) 245 (245-245) NA
.029 47 (32-62) 22 (1-43)
NA NA NA
NA NA
NA NA NA
.265 35 (26-44) 51 (23-80)
NA NA NA
.687 45 (28-61) 33 (8-58)
.225 69 (57-81) 245 (245-245)
.053 46 (31-62) 24 (1-46)
.515 187 (56-317) 68 (44-93)
.428 47 (28-65) 33 (14-51)
.937 75 (70-80) 193 (69-317)
.097 46 (30-61) 26 (8-44)
.515 187 (56-317) 68 (44-93)
.097 46 (30-61) 26 (8-44)
.515 187 (56-317) 68 (44-93)
NA NA NA
NA NA NA
NA NA NA
NA NA NA .061
.034 45 (31-58) 18 (0-51)
201 (97-305) 53 (32-73) .261
30 (18-43) 44 (26-61)
.027 60 (35-86) 245 (245-245)
.695 35 (25-44) 42 (21-63)
.643 72 (72-72) 203 (100-306)
NA NA NA
NA NA NA
NA, not applicable; S, stromal; T, tumor. For definitions of other abbreviations, see Table 1. a Bold values refer to statistical significance (P < .05).
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© American Society for Clinical Pathology
AJCP / Original Article
A
B
1.0 Cumulative Disease-Free Survival
Cumulative Disease-Free Survival
1.0
0.8
0.6 MCT4 (T) (–)
0.4
0.2 MCT4 (T) (+)
0.0
0.8
0.6 PSPH (T) (–)
0.4
0.2 PSPH (T) (+)
0.0 0
20
40
60
80
0
Months
C
Cumulative Overall Survival
1.0
20
40
60
80
Months
❚Figure 2❚ Kaplan-Meier curves for disease-free survival and overall survival of lacrimal gland adenoid cystic carcinoma correlated with expression of the metabolism-related proteins. Factors associated with a shorter disease-free survival were monocarboxylate transporter 4 (MCT4) positivity (P = .029) (A) and phosphoserine phosphatase (PSPH) positivity (P = .034) (B). The factor associated with shorter overall survival was serine hydroxy methyltransferase 1 (SHMT1) negativity (P = .027) (C). T, tumor.
SHMT1 (T) (+)
0.8
0.6
0.4
0.2 SHMT1 (T) (–)
0.0 0
50
100
150
200
250
Months
❚Table 6❚ Multivariate Analyses of Disease-Free Survival and Overall Survival in Lacrimal Gland Adenoid Cystic Carcinoma Disease-Free Survival
Overall Survival
Included Parameter
Hazard Ratio (95% CI)
P Value
Hazard Ratio (95% CI)
P Value
Histologic grade: 1/2 vs 3 Lymphovascular invasion: absent vs present MCT4 (T): negative vs positive PSPH (T): negative vs positive SHMT1 (T): negative vs positive
0.277 (0.023-3.362) 0.000 3.842 (0.308-47.86) 17.86 (0.970-328.8) Not included
.313 .976 .296 .052
5.120 (0.000-1.184) 3.411 (0.000-5.859) Not included 242.9 (0.000-6.904) 414.2 (0.000-6.494)
.923 .949 .854 .697
MCT4, monocarboxylate transporter 4; PSPH, phosphoserine phosphatase; S, stromal; SHMT1, serine hydroxy methyltransferase 1; T, tumor.
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20. van den Heuvel AP, Jing J, Wooster RF, et al. Analysis of glutamine dependency in non-small cell lung cancer: GLS1 splice variant GAC is essential for cancer cell growth. Cancer Biol Ther. 2012;13:1185-1194. 21. von Holstein SL, Fehr A, Persson M, et al. Adenoid cystic carcinoma of the lacrimal gland: MYB gene activation, genomic imbalances, and clinical characteristics. Ophthalmology. 2013;120:2130-2138. 22. Mitani Y, Li J, Rao PH, et al. Comprehensive analysis of the MYB-NFIB gene fusion in salivary adenoid cystic carcinoma: Incidence, variability, and clinicopathologic significance. Clin Cancer Res. 2010;16:4722-4731. 23. Gomez-Maldonado J, Avila C, Torre F, et al. Functional interactions between a glutamine synthetase promoter and MYB proteins. Plant J. 2004;39:513-526. 24. Bonfitto VL, Demasi AP, Costa AF, et al. High-grade transformation of adenoid cystic carcinomas: a study of the expression of GLUT1 glucose transporter and of mitochondrial antigen. J Clin Pathol. 2010;63:615-619. 25. Papandreou I, Cairns RA, Fontana L, et al. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab. 2006;3:187-197. 26. Fisel P, Kruck S, Winter S, et al. DNA methylation of the SLC16A3 promoter regulates expression of the human lactate transporter MCT4 in renal cancer with consequences for clinical outcome. Clin Cancer Res. 2013;19:5170-5181. 27. Gotanda Y, Akagi Y, Kawahara A, et al. Expression of monocarboxylate transporter (MCT)-4 in colorectal cancer and its role: MCT4 contributes to the growth of colorectal cancer with vascular endothelial growth factor. Anticancer Res. 2013;33:2941-2947. 28. Nakayama Y, Torigoe T, Inoue Y, et al. Prognostic significance of monocarboxylate transporter 4 expression in patients with colorectal cancer. Exp Ther Med. 2012;3:25-30. 29. Witkiewicz AK, Whitaker-Menezes D, Dasgupta A, et al. Using the “reverse Warburg effect” to identify high-risk breast cancer patients: stromal MCT4 predicts poor clinical outcome in triple-negative breast cancers. Cell Cycle. 2012;11:1108-1117. 30. Meijer TW, Schuurbiers OC, Kaanders JH, et al. Differences in metabolism between adeno- and squamous cell non-small cell lung carcinomas: spatial distribution and prognostic value of GLUT1 and MCT4. Lung Cancer. 2012;76:316-323. 31. Kuo CH, Miyazaki D, Nawata N, et al. Prognosis-determinant candidate genes identified by whole genome scanning in eyes with pterygia. Invest Ophthalmol Vis Sci. 2007;48:3566-3575. 32. Budai B, Komlosi V, Adleff V, et al. Impact of SHMT1 polymorphism on the clinical outcome of patients with metastatic colorectal cancer treated with firstline FOLFIRI+bevacizumab. Pharmacogenet Genomics. 2012;22:69-72. 33. Morris DI, Robbins JD, Ruoho AE, et al. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter. J Biol Chem. 1991;266:13377-13384. 34. Possemato R, Marks KM, Shaul YD, et al. Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature. 2011;476:346-350. 35. Yang C, Sudderth J, Dang T, et al. Glioblastoma cells require glutamate dehydrogenase to survive impairments of glucose metabolism or Akt signaling. Cancer Res. 2009;69:7986-7993.
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Expression of Metabolism-Related Proteins in Lacrimal Gland Adenoid Cystic Carcinoma Ja Seung Koo, MD, PhD,1 and Jin Sook Yoon, MD, PhD2 From the 1Department of Pathology and 2Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, South Korea. Key Words: Adenoid cystic carcinoma; Lacrimal gland; Metabolism-related protein; Protein expression; Salivary gland Am J Clin Pathol April 2015;143:584-592 DOI: 10.1309/AJCPXAYH10WENLTC
ABSTRACT Objectives: To investigate the expression and the clinical implications of metabolism-related proteins in lacrimal gland adenoid cystic carcinoma (ACC) in comparison with salivary gland ACC. Methods: Human tissue samples of lacrimal gland ACC (n = 11) and salivary gland ACC (n = 64) were analyzed. Immunochemistry was used to measure expression of proteins related to glycolysis (glucose transporter 1, hexokinase II, carbonic anhydrase IX, and monocarboxylate transporter 4 [MCT4]), glutaminolysis (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter 2 [ASCT2]), mitochondria (adenosine triphosphate [ATP] synthase, succinate dehydrogenase A [SDHA], and succinate dehydrogenase B), and glycolytic intermediate metabolism (phosphoserine phosphatase [PSPH], serine hydroxymethyl transferase 1 [SHMT1]). Results: GLS1 and ASCT2 were more highly expressed, and GDH, ATP synthase, and SDHA were expressed to a lesser degree in lacrimal gland ACC than in salivary gland ACC (P < .05). Lacrimal gland ACC showed less of a mitochondrial phenotype than did salivary gland ACC (P = .001). Positivity of MCT4 and PSPH was related to shorter disease-free survival, and SHMT1 was related to shorter overall survival (P < .05). Conclusions: Lacrimal gland ACC exhibited higher expression of GLS1 and ASCT2, compared with salivary gland ACC. Overexpression of MCT4, PSPH, and SHMT1 was associated with poorer prognosis.
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Adenoid cystic carcinoma (ACC) is a malignancy of secretory glands, including the lacrimal and salivary glands, with a poor prognosis and difficult decisions for treatment.1 Despite a slow rate of growth, lacrimal gland ACCs are highly aggressive and prone to local recurrence and perineural spread to the cranial nerves.1 ACC represents the most common malignancy of the lacrimal glands, comprising 3.8% of primary ocular adnexal tumors.2 ACC accounts for approximately 40% of salivary gland malignancies and is characterized by a slow but persistent progression, with multiple local recurrence and metastasis to the lung, bone, and brain occurring in approximately 50% of patients.3,4 There are only a few studies of lacrimal gland ACC, in comparison with salivary gland ACC, due to rarity of the tumor. Factors related to poor prognosis of salivary gland ACC include advanced stage, solid architecture, high histologic grade, perineural invasion, and positive surgical resection margin.5-7 Compared with salivary gland ACC, lacrimal gland ACC has shown clinical features of younger age and poorer prognosis.8 The causes of poorer prognosis of lacrimal gland ACC include the high incomplete excision rate due to complex orbital anatomy, invasion to nearby structures, and subsequent metastases.1,9 Primary treatments of lacrimal gland ACC are en bloc surgical excision and postoperative radiation, as needed.10 Tse et al11 recently reported that neoadjuvant intra-arterial cytoreductive chemotherapy improved overall survival and decreased recurrence in 19 lacrimal gland ACCs. However, controversy regarding treatment still exists.12 Neutron radiation therapy achieved 80% of 5-year local control in 11 cases, although late recurrence and distant metastasis remained as challenges.13 There is no effective treatment
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❚Table 1❚ Clone Source, Dilutions Used, Sources of Antibodies Used, and Expression Pattern Antibody Glycolysis related Glut1 Hexokinase II CAIX MCT4 Glutaminolysis related GLS1 GDH ASCT2 Mitochondrial related ATP synthase SDHA SDHB Serine/glycine metabolism related PHGDH PSAT1 PSPH SHMT1 GLDC
Clone
Dilution Source
Expression Pattern
SPM498 Polyclonal Polyclonal Polyclonal
1:200 1: 100 1:100 1:100
Abcam, Cambridge, England Abcam Abcam Santa Cruz Biotech, Santa Cruz, CA
Cytoplasmic and/or membranous Cytoplasmic Cytoplasmic Membranous
Polyclonal Polyclonal Polyclonal
1:50 1:100 1:100
Abcam Abcam Abcam
Cytoplasmic Cytoplasmic Membranous
15H4C4 1:100 2E3GC12FB2AE2 1:100 21A11AE7 1:100
Abcam Abcam Abcam
Cytoplasmic Cytoplasmic Cytoplasmic
Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal
Abcam Abcam Abcam Abcam Abcam
Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic
1:100 1:100 1:100 1:100 1:100
ASCT2, amino acid transporter 2; ATP, adenosine triphosphate; CAIX, carbonic anhydrase IX; GDH, glutamate dehydrogenase; GLDC, glycine dehydrogenase, decarboxylating; GLS1, glutaminase 1; Glut1, glucose transporter 1; MCT4, monocarboxylate transporter 4; PHGDH, phosphoglycerate dehydrogenase; PSAT1, phosphohydroxythreonine aminotransferase 1; PSPH, phosphoserine phosphatase; SDHA, succinate dehydrogenase A; SDHB, succinate dehydrogenase B; SHMT1, serine hydroxy methyltransferase 1.
modality in cases of recurrence or metastasis of lacrimal gland ACC. New treatments targeting this rare and lifethreatening cancer are needed. One of the hallmarks of cancer is metabolic deregulation, which has been recently discussed and investigated as a possible therapeutic target to treat cancer.14 Many tumors display increased glucose uptake and metabolism through the process of aerobic glycolysis, also known as the Warburg effect, suggesting that glycolysis is a major component of tumor metabolism.15 However, together with glycolysis, cancer cells use several metabolic systems, including glutaminolysis, mitochondria metabolism, and glycolytic intermediate metabolism.16,17 Thus far, no studies have investigated the characteristics of metabolism in lacrimal gland ACC. The purpose of this study was to demonstrate the expression of proteins related to several metabolic pathways, including glycolysis, glutaminolysis, mitochondrial metabolism, and glycolytic intermediate metabolism in lacrimal gland ACC in comparison with salivary gland ACC, and to find prognostic factors associated with survival.
Materials and Methods Patient Selection and Clinicopathologic Evaluation Formalin-fixed, paraffin-embedded tissue samples of lacrimal gland ACC, collected from January 1997 through December 2012, at Severance Hospital in Seoul, South Korea, were used for analyses. The study was approved by
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the Institutional Review Board of Severance Hospital and adhered to the tenets of the Declaration of Helsinki. Clinical information such as age at surgery, sex, tumor side, symptoms, and visual acuity were informed from medical chart recordings. Tumor stage classifications followed the seventh American Joint Committee on Cancer staging system, and histologic features of H&E slides were reviewed by a specialized pathologist (J.S.K.). Histologic grading of tumors followed the indications established by Szanto et al18 as follows: grade I, no solid component; grade II, ACC with less than 30% solid component; and grade III, ACC with more than 30% solid component. Histologic type was determined by predominant morphologic growth patterns and divided into cribriform, tubular, and solid patterns. Perineural invasion, tumor margin (expanding, infiltrative), and tumor involvement in the surgical resection margin were evaluated. As a control group, 64 cases of major salivary gland ACC were included in the same periods of tissue collection. Immunohistochemistry The antibodies used for immunohistochemistry (IHC) in this study are listed in ❚Table 1❚. IHC was performed using the Ventana Discovery XT automated staining system (Ventana Medical Systems, Tucson, AZ) following the manufacturer’s protocols. Briefly, Slides of 3-µm–thick tissue sections were incubated for 1 hour at room temperature in standard Cell Conditioning 1 buffer (citrate buffer, pH 6.0; Ventana Medical Systems). The tissue samples were subsequently incubated with primary antibodies for 32 minutes at room temperature, washed, and incubated with the
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❚Table 2❚ Clinicopathologic Characteristics of Lacrimal Gland Adenoid Cystic Carcinoma Age, Case No. y/Sex
Tumor Size, Main Histologic Histologic cm/Tumor Side Type Grade
Lymphovascular Ocular Invasion Symptoms
Local Chemotherapy/ Recurrence/ Radiation Metastasis Survival Therapy
1 2 3 4 5 6 7 8 9 10 11
3.0/R 2.6/L 4.0/L 3.5/R 3.5/R 3.0/R 2.5/L 3.6/R 3.0/R 3.5/L 2.5/R
No No No No No No Yes No Yes No No
Yes/yes No/yes Yes/yes No/yes No/no Yes/no No/no No/yes No/no Yes/yes Yes/yes
41/F 21/M 63/F 54/M 28/M 72/M 57/M 51/M 35/M 61/F 43/M
Cribriform Cribriform Solid Solid Tubular Solid Cribriform Tubular Cribriform Tubular Cribriform
2 1 3 3 1 3 1 2 1 2 1
ultraView Universal DAB kit (Ventana Medical Systems) for 16 minutes at room temperature. Then, tissue sections were stained with Hematoxylin ReaDi solution (Ventana Medical Systems). IHC included the appropriate positive and negative controls. IHC was performed to measure expression of proteins related to glycolysis (glucose transporter 1 [Glut1], hexokinase II, carbonic anhydrase IX [CAIX], and monocarboxylate transporter 4 [MCT4]), glutaminolysis (glutaminase 1 [GLS1] and glutamate dehydrogenase [GDH]), amino acid transporter 2 (ASCT2), mitochondrial metabolism (adenosine triphosphate [ATP] synthase, succinate dehydrogenase A [SDHA], and succinate dehydrogenase B [SDHB]), and glycolytic intermediate metabolism (phosphoglycerate dehydrogenase [PHGDH], phosphohydroxythreonine aminotransferase 1 [PSAT1], phosphoserine phosphatase [PSPH], serine hydroxymethyl transferase 1 [SHMT1], and glycine dehydrogenase decarboxylase [GLDC]) in 11 cases of lacrimal gland ACC and 64 cases of major salivary gland ACC. Interpretation of Immunohistochemical Results Results of IHC were defined as the proportion of stained cells × immunostaining intensity. The proportion of stained cell was defined as follows: 0, negative; 1, less than 30% positivity; and 2, 30% or more positivity. Immunostaining intensity was defined as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The proportion of stained cells × immunostaining intensity was defined as follows: 0 to 1 was negative, and 2 to 6 was positive.19 Classification of Tumor Metabolic Subtype Tumor metabolic subtype was classified according to the expression pattern of metabolism-related proteins: (1) glycolysis type (more than three positive expressions in Glut1, hexokinase II, CAIX, and MCT4) and nonglycolysis type, (2) glutaminolysis type (more than two positive expressions in GLS1, GDH, and ASCT2) and nonglutaminolysis
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Proptosis Proptosis Lid swelling Proptosis Proptosis Proptosis Palpable mass Proptosis Proptosis Proptosis Lid swelling
Dead Alive Dead Dead Alive Alive Alive Alive Alive Alive Alive
No/yes No/yes No/no No/yes Yes/yes Yes/yes No/yes Yes/yes Yes/yes Yes/yes Yes/yes
type, (3) mitochondrial type (more than two positive expressions in ATP synthase, SDHA, and SDHB) and nonmitochondrial type, and (4) serine/glycine type (more than three positive expressions in PHGDH, PSAT1, PSPH, SHMT1, and GLDC) and nonserine/glycine type. Statistical Analysis Data were statistically processed using SPSS for Windows version 12.0 (SPSS, Chicago, IL). The Student t test and Fisher exact test were used for continuous and categorical variables, respectively. To analyze data with multiple comparisons, a corrected P value with application of the Bonferroni method for multiple comparisons was used. Statistical significance was defined as P < .05. Kaplan-Meier survival curves and log-rank statistics were employed to evaluate survival time and time to tumor metastasis, respectively. Multivariate regression analysis was performed using the Cox proportional hazards model.
Results Basal Characteristics of Lacrimal Gland ACC Initially, we found that 14 patients received an operation for lacrimal gland ACC in our database, but since the histologic analysis of the tissue block from three patients was impossible, this study included 11 cases of lacrimal gland ACC ❚Table 2❚. Clinical characteristics included age from 21 to 72 years, with three women and eight men. The size of the tumor was 2.5 to 4.0 cm, and the main histologic type was cribriform (n = 5). The most common histologic grade was grade 1 (n = 5). Perineural invasion was not found in all cases. Lymphovascular invasion was observed in two cases, local recurrence in five cases, and distant metastases in seven cases (brain, n = 5; lung, n = 2). Three patients died of disease.
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❚Table 3❚ Comparisons of the Expression of Metabolism-Related Proteins Between Lacrimal Gland and Salivary Gland Adenoid Cystic Carcinoma (ACC)a Parameter
Total (n = 75)
Lacrimal Salivary Gland ACC Gland ACC (n = 11) (n = 64)
P Value
Glut-1 (T) Hexokinase II (T) CAIX (T) MCT4 (T) MCT4 (S) GLS1 (T) GDH (T) ASCT2 (T) ATP synthase (T) SDHA (T) SDHB (T) PHGDH (T) PSAT1 (T) PSPH (T) SHMT1 (T) SHMT1 (S) GLDC (T)
27 (36.0) 7 (9.3) 17 (22.7) 14 (18.7) 9 (12.0) 15 (20.0) 64 (85.3) 6 (8.0) 63 (84.0) 60 (80.0) 39 (52.0) 66 (88.0) 9 (12.0) 4 (5.3) 65 (86.7) 26 (34.7) 7 (9.3)
6 (54.5) 2 (18.2) 3 (27.3) 3 (27.3) 1 (9.1) 5 (45.5) 4 (36.4) 3 (27.3) 5 (45.5) 4 (36.4) 4 (36.4) 11 (100.0) 0 (0.0) 2 (18.2) 8 (72.7) 6 (54.5) 0 (0.0)
.188 .272 .704 .420 1.000 .037 3 cm
❚Figure 1❚ Correlations between metabolism-related proteins and clinicopathologic factors in lacrimal gland adenoid cystic carcinoma. All cases with tumor sizes 3 cm or less (n = 6) were adenosine triphosphate (ATP) synthase negative, whereas all cases with tumor size more than 3 cm (n = 5) expressed more ATP synthase.
addition, this study could not demonstrate many significant findings despite numerous analyses. Additional studies are warranted to confirm the different expression of these proteins between lacrimal gland ACC and salivary gland ACC.
Conclusions We have demonstrated for the first time that lacrimal gland ACC showed distinct profiles of several metabolismrelated protein expression. Compared with salivary gland ACC, lacrimal gland ACC exhibited lower expression of GDH, ATP synthase, and SDHA and higher expression of ASCT2 and GLS1. Metabolic regulatory proteins have emerged as attractive targets for cancer therapy. For example, clinical and preclinical studies of inhibitors of Glut1,33 PHGDH,34 and GDH35 have been evaluated. Our data may provide further insights and a basis for further studies to identify potential cancer targets from among the metabolism-related proteins in lacrimal gland ACC. Because lacrimal gland ACC is fatal, has a poor prognosis, and is without available and effective chemotherapeutic agents, identification of novel therapeutic drugs targeting these metabolic regulators in lacrimal gland ACC is essential. Address reprint requests to Dr Yoon: Dept of Ophthalmology, Institute of Vision Research, Yonsei University College of Medicine, Severance Hospital, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, South Korea; email: [email protected]. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2012R1A1A1002886). The funding organization had no role in the design or conduct of this research.
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❚Table 5❚ Univariate Analyses by Log-Rank Tests of the Impact of Metabolism-Related Proteins on Disease-Free Survival and Overall Survival in Lacrimal Gland Adenoid Cystic Carcinomaa Disease-Free Survival Parameter Glut-1 (T) Negative Positive Hexokinase II (T) Negative Positive CAIX (T) Negative Positive MCT4 (T) Negative Positive MCT4 (S) Negative Positive GLS1 (T) Negative Positive GDH (T) Negative Positive ASCT2 (T) Negative Positive ATP synthase (T) Negative Positive SDHA (T) Negative Positive SDHB (T) Negative Positive PHGDH (T) Negative Positive PSAT1 (T) Negative Positive PSPH (T) Negative Positive SHMT1 (T) Negative Positive SHMT1 (S) Negative Positive GLDC (T) Negative Positive
No. of Months (95% CI)
P Value
Overall Survival No. of Months (95% CI)
.929 41 (24-59) 36 (18-53)
P Value .180
68 (54-81) 245 (245-245) .977
41 (24-58) 44 (26-61)
.379 80 (73-86) 141 (0-344)
.390 46 (31-62) 28 (0-57)
.431 74 (61-86) 245 (245-245) NA
.029 47 (32-62) 22 (1-43)
NA NA NA
NA NA
NA NA NA
.265 35 (26-44) 51 (23-80)
NA NA NA
.687 45 (28-61) 33 (8-58)
.225 69 (57-81) 245 (245-245)
.053 46 (31-62) 24 (1-46)
.515 187 (56-317) 68 (44-93)
.428 47 (28-65) 33 (14-51)
.937 75 (70-80) 193 (69-317)
.097 46 (30-61) 26 (8-44)
.515 187 (56-317) 68 (44-93)
.097 46 (30-61) 26 (8-44)
.515 187 (56-317) 68 (44-93)
NA NA NA
NA NA NA
NA NA NA
NA NA NA .061
.034 45 (31-58) 18 (0-51)
201 (97-305) 53 (32-73) .261
30 (18-43) 44 (26-61)
.027 60 (35-86) 245 (245-245)
.695 35 (25-44) 42 (21-63)
.643 72 (72-72) 203 (100-306)
NA NA NA
NA NA NA
NA, not applicable; S, stromal; T, tumor. For definitions of other abbreviations, see Table 1. a Bold values refer to statistical significance (P < .05).
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A
B
1.0 Cumulative Disease-Free Survival
Cumulative Disease-Free Survival
1.0
0.8
0.6 MCT4 (T) (–)
0.4
0.2 MCT4 (T) (+)
0.0
0.8
0.6 PSPH (T) (–)
0.4
0.2 PSPH (T) (+)
0.0 0
20
40
60
80
0
Months
C
Cumulative Overall Survival
1.0
20
40
60
80
Months
❚Figure 2❚ Kaplan-Meier curves for disease-free survival and overall survival of lacrimal gland adenoid cystic carcinoma correlated with expression of the metabolism-related proteins. Factors associated with a shorter disease-free survival were monocarboxylate transporter 4 (MCT4) positivity (P = .029) (A) and phosphoserine phosphatase (PSPH) positivity (P = .034) (B). The factor associated with shorter overall survival was serine hydroxy methyltransferase 1 (SHMT1) negativity (P = .027) (C). T, tumor.
SHMT1 (T) (+)
0.8
0.6
0.4
0.2 SHMT1 (T) (–)
0.0 0
50
100
150
200
250
Months
❚Table 6❚ Multivariate Analyses of Disease-Free Survival and Overall Survival in Lacrimal Gland Adenoid Cystic Carcinoma Disease-Free Survival
Overall Survival
Included Parameter
Hazard Ratio (95% CI)
P Value
Hazard Ratio (95% CI)
P Value
Histologic grade: 1/2 vs 3 Lymphovascular invasion: absent vs present MCT4 (T): negative vs positive PSPH (T): negative vs positive SHMT1 (T): negative vs positive
0.277 (0.023-3.362) 0.000 3.842 (0.308-47.86) 17.86 (0.970-328.8) Not included
.313 .976 .296 .052
5.120 (0.000-1.184) 3.411 (0.000-5.859) Not included 242.9 (0.000-6.904) 414.2 (0.000-6.494)
.923 .949 .854 .697
MCT4, monocarboxylate transporter 4; PSPH, phosphoserine phosphatase; S, stromal; SHMT1, serine hydroxy methyltransferase 1; T, tumor.
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![[Adenoid cystic carcinoma of the lacrimal gland].](/assets/img/hold.png)
