Acta Oto-Laryngologica. 2014; 134: 536–542
ORIGINAL ARTICLE
Galectin-1 overexpression in nasopharyngeal carcinoma: effect on survival
SHIH-LUN CHANG1,4, CHIEN-FENG LI2, CHARLENE LIN3 & YUNG-SONG LIN1,5 1
Department of Otolaryngology and, 2Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan, 3Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA, 4Department of Otolaryngology, School of Medicine, Medical College, Kaohsiung Medical University, Kaohsiung and 5Department of Otolaryngology, School of Medicine, Medical College, Taipei Medical University, Taipei, Taiwan
Abstract Conclusions: Galectin-1 overexpression is significantly correlated with the survival rate of patients with nasopharyngeal carcinoma (NPC). Immunohistochemical analysis of galectin-1 expression might be useful for identifying patients with a high risk of distant metastasis and for prompting timely adjuvant systemic therapy for patients with aggressive NPC. Objectives: We examined the effect of galectin-1 on the survival rate of patients with NPC. Methods: A total of 124 patients diagnosed between 1998 and 2002 with NPC without distant metastasis were enrolled in this single-center historical prospective cohort study. Immunohistochemical analysis was used to correlate the galectin-1 expression score in the cytoplasm and the survival rate of patients with NPC. Results: Patients with NPC who overexpressed galectin-1 in cytoplasm showed more aggressive tumor growth and significantly shorter disease-specific survival (DSS) (p = 0.0037) and distant metastasis-free survival (DMFS) (p = 0.0006) than patients with NPC who did not overexpress galectin-1. Multivariate analysis showed that galectin-1 overexpression remained prognostically independent for DSS (p = 0.031, hazard ratio = 1.821), and DMFS (p = 0.005, hazard ratio = 2.417), together with the advanced III–IV stages.
Keywords: Survival rate, head and neck cancer
Introduction Galectin-1 is one of the -galactoside-binding proteins that display biologically diverse activities in the pathogenesis of inflammation and cancer. This protein is commonly found and secreted at high levels in cancer cells [1]. Galectin-1 expression and overexpression in tumors and the tissue surrounding the tumor have been considered a sign of malignant tumor progression that is often related to distant metastasis, local invasion, and tumor immune-escape [2]. The potential role of galectin-1 in the acquisition of immune privilege by various types of tumors has been suggested [3], including for head and neck malignancies [4]. Galectins as modulators of tumor progression in head and neck squamous cell carcinomas are involved in
controlling cell proliferation, cell death, and cell migration, and in modulating various functions of the immune system [5]. The hypothesis of tumorimmune privilege has been experimentally supported [6]. In recent years, several studies [3-5,7,8] have discussed the effect of galectin-1 in head and neck malignancies. However, to our knowledge, there is no study in the English literature that has directly examined the effect of galectin-1 expression on the survival rate of patients with nasopharyngeal carcinoma (NPC). Because there are many types of head and neck malignancies, it is impossible that a single biomarker can predict the outcome of any individual malignancy. For example, NPC is a distinctive type of head and neck malignancy prevalent in people who live in Southeast Asia, and is now ranked the ninth
Correspondence: Yung-Song Lin MD, Department of Otolaryngology, School of Medicine, Medical College, Taipei Medical University, 250 Wu-Hsing Street, Taipei City, 110, Taiwan. E-mail: [email protected]
(Received 19 September 2013; accepted 13 November 2013) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2013.868603
Galectin-1 and nasopharyngeal carcinoma most common cancer in Taiwan, with an approximate incidence rate of 9.99 per 100 000 in males and 2.98 per 100 000 in females [9]. Thus, early diagnosis, exact histologic grading, and accurate prediction are critical for developing better treatment options and improving the prognosis of NPC. A future research goal might be based upon finding novel prognostic factors to precisely predict the biological behavior of the tumors. Hence, we conducted a cohort study to examine the effect of galectin-1 expression on the survival rate of patients with NPC. An examination of the amount of galectin-1 expression in NPC will, hopefully, provide useful information to physicians for selecting the optimal therapy, and to researchers working to identify the potentials of immunotherapy for patients with NPC. Material and methods Patients and tumor specimens The Chi Mei Medical Center institutional review board approved procurement of formalin-fixed NPC tissue for this study. Available paraffin-embedded tissue blocks were retrieved from 124 patients with NPC who had undergone biopsies between January 1998 and December 2002 during their initial diagnosis. None of the patients had distant metastasis at the time of diagnosis. Two pathologists reappraised the histological subtypes according to the current WHO classification. The tumor staging was reappraised using the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/ AJCC) staging system [10]. Cell culture and immunoblotting analysis Dysplastic oral keratinocyte (DOK) and NPCderived HONE-1 and TW01 cell lines were maintained in Dulbecco’s modified Eagle’s medium/ Hams-F12 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Biological Industry, Kibbutz Beit Haemek, Israel) in a 37 C, 5% CO2 incubator. Western blot assays were performed to evaluate the expression levels of galectin-1. Equal amounts of total protein (25 mg) extracted from the cell lines under various conditions were separated on 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel NuPAGE (Invitrogen), and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK). After they had been blocked with 5% skimmed milk in TBST (Tris-buffered saline and Tween 20) buffer at room temperature for 1 h, the membranes
537
were probed with antibodies at 48 C overnight against galectin-1 (1:1000) (cat. no. 437400; Invitrogen) and GADPH (glyceraldehyde-3-phosphate dehydrogenase) as a loading control (Clone 6C5, 1:10 000; Millipore, Beverly, MA, USA). After they had been incubated with the secondary antibody at room temperature for 1.5 h, protein expression was detected using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences). Immunohistochemical staining and assessment of galectin-1 expression Tissue sections 3 m thick were cut from paraffinembedded blocks. Slides were deparaffinized with xylene, rehydrated with ethanol, and heated in a microwave oven for 7 min to retrieve antigen epitopes in a 10 mM citrate buffer (pH 6). Endogenous peroxidase was quenched using 3% H2O2 treatment. Slides were washed with Tris buffered saline for 15 min, and then incubated with primary monoclonal antibodies targeting Gal-1 (1:200) (cat. no. 437400; Invitrogen) for 1 h. Primary antibodies were detected using a peroxidase-based kit (ChemMate EnVision kit K5007; DAKO, Carpinteria, CA, USA). The slides were incubated with secondary antibodies for 30 min, developed with 3,3-diaminobenzidine for 5 min, and then counterstained with hematoxylin. A mammary invasive ductal carcinoma cell line known to express galectin-1 was used as a positive control. Rabbit serum IgG was used to replace primary antibody as a negative control. Without prior knowledge of the patients’ tumor stages and the outcomes of their treatment, two pathologists (H.Y.H and C.F.L.) used a multi-headed microscope to score galectin-1 immunoexpression to arrive at a consensus. Galectin1 staining is interpreted using the immunohistochemistry H-score: H = (percentage score) (intensity score + 1). The percentage score ranged from 0 to 4, based on the proportion of nuclear-stained tumor cells (0, 50%). The intensity score ranged from 0 to 3 (0, negative staining; 1, weak nuclear staining; 2, moderate nuclear staining; and 3, strong nuclear staining), as described previously [11]. Treatment and follow-up All 124 patients were regularly monitored after radiotherapy until death or their last appointment. The mean follow-up duration was 67.0 months (range 3–141). Seven patients were not available for image studies after therapy, but the remaining 117 patients underwent a complete course of radiotherapy (total dose ‡ 7000 cGy) and also cisplatin-based chemotherapy if they reached
538
S.-L. Chang et al. DOK
stages II–IV of NPC, in accordance with the published protocol. The WHO reported a set of criteria that include completing clinical examinations and imaging modalities to assess treatment responses. Ninety-four patients met all the criteria, and seven patients met some of the same criteria.
GAPDH
Figure 1. Immunoblotting analyses showed that the galectin-1 protein level was not detected in dysplastic oral keratinocyte (DOK) cells. However, it was expressed in nasopharyngeal carcinoma (NPC)-derived HONE and TW01 cells.
Prognostic impact of galectin-1 expression In a mean follow-up of 67.0 months (range 3–141), local recurrence, distant metastasis, and tumorassociated mortality developed with a median duration of 21, 21, and 33 months, respectively (Table II). With respect to the three end point analyses, T3– 4 status, N2–3 status, and stages III and IV at diagnosis were all significant predictors of disease Table I. Associations between galectin-1 expression and other important clinicopathologic variables.
Results Galectin-1 was highly expressed in HONE-1 and TW01 cells Galectin-1 protein was not detected in DOK1 cells. However, it was highly expressed in HONE-1 and TW01 cells lines (Figure 1), which suggests that galectin-1 is involved in the carcinogenesis of NPC. Immunohistochemical expression of galectin-1 and its associations with clinicopathological variables The 124 patients with NPC (95 men, 29 women) had a mean age of 48.6 years (range 20–83) (Table I). Seven patients were classified as stage I, 31 as stage II, 46 as stage III, and 40 as stage IV. Five patients had keratinizing squamous cell carcinoma, 54 had differentiated non-keratinizing carcinoma, and 65 had undifferentiated non-keratinizing carcinoma. Although galectin-1 immunoexpression was rarely detected in the non-neoplastic nasopharyngeal mucosal epithelia, it was expressed in notable amounts in a certain number of cases of NPC with a cytoplasmic pattern (Figure 2). A higher level of galectin-1 expression was associated with a more advanced primary tumor classification (p = 0.009) and a more advanced AJCC stage (III–IV; p = 0.019).
TWO1
Galectin-1
Statistical analysis SPSS 14 for Windows was used for all statistical analyses. The associations of galectin-1 expression status with various clinicopathological parameters were evaluated using a 2 or Fisher’s exact test. The end point analyses were local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), and disease-specific survival (DSS), all of which were calculated from the starting date of radiotherapy to the date of death or the last appointment. Patients who stopped attending their follow-up appointments were censored on the latest follow-up date. Survival curves were plotted using the Kaplan–Meier method, and log-rank tests were done to evaluate prognostic differences between the groups. Multivariate analysis was done using the Cox proportional hazards model. For all analyses, two-sided tests of significance were used; significance was set at p < 0.05.
HONE
Galectin-1 expression Characteristic
Low
High
p value
Male
46
49
0.524
Female
16
13
ORIGINAL ARTICLE
Galectin-1 overexpression in nasopharyngeal carcinoma: effect on survival
SHIH-LUN CHANG1,4, CHIEN-FENG LI2, CHARLENE LIN3 & YUNG-SONG LIN1,5 1
Department of Otolaryngology and, 2Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan, 3Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA, 4Department of Otolaryngology, School of Medicine, Medical College, Kaohsiung Medical University, Kaohsiung and 5Department of Otolaryngology, School of Medicine, Medical College, Taipei Medical University, Taipei, Taiwan
Abstract Conclusions: Galectin-1 overexpression is significantly correlated with the survival rate of patients with nasopharyngeal carcinoma (NPC). Immunohistochemical analysis of galectin-1 expression might be useful for identifying patients with a high risk of distant metastasis and for prompting timely adjuvant systemic therapy for patients with aggressive NPC. Objectives: We examined the effect of galectin-1 on the survival rate of patients with NPC. Methods: A total of 124 patients diagnosed between 1998 and 2002 with NPC without distant metastasis were enrolled in this single-center historical prospective cohort study. Immunohistochemical analysis was used to correlate the galectin-1 expression score in the cytoplasm and the survival rate of patients with NPC. Results: Patients with NPC who overexpressed galectin-1 in cytoplasm showed more aggressive tumor growth and significantly shorter disease-specific survival (DSS) (p = 0.0037) and distant metastasis-free survival (DMFS) (p = 0.0006) than patients with NPC who did not overexpress galectin-1. Multivariate analysis showed that galectin-1 overexpression remained prognostically independent for DSS (p = 0.031, hazard ratio = 1.821), and DMFS (p = 0.005, hazard ratio = 2.417), together with the advanced III–IV stages.
Keywords: Survival rate, head and neck cancer
Introduction Galectin-1 is one of the -galactoside-binding proteins that display biologically diverse activities in the pathogenesis of inflammation and cancer. This protein is commonly found and secreted at high levels in cancer cells [1]. Galectin-1 expression and overexpression in tumors and the tissue surrounding the tumor have been considered a sign of malignant tumor progression that is often related to distant metastasis, local invasion, and tumor immune-escape [2]. The potential role of galectin-1 in the acquisition of immune privilege by various types of tumors has been suggested [3], including for head and neck malignancies [4]. Galectins as modulators of tumor progression in head and neck squamous cell carcinomas are involved in
controlling cell proliferation, cell death, and cell migration, and in modulating various functions of the immune system [5]. The hypothesis of tumorimmune privilege has been experimentally supported [6]. In recent years, several studies [3-5,7,8] have discussed the effect of galectin-1 in head and neck malignancies. However, to our knowledge, there is no study in the English literature that has directly examined the effect of galectin-1 expression on the survival rate of patients with nasopharyngeal carcinoma (NPC). Because there are many types of head and neck malignancies, it is impossible that a single biomarker can predict the outcome of any individual malignancy. For example, NPC is a distinctive type of head and neck malignancy prevalent in people who live in Southeast Asia, and is now ranked the ninth
Correspondence: Yung-Song Lin MD, Department of Otolaryngology, School of Medicine, Medical College, Taipei Medical University, 250 Wu-Hsing Street, Taipei City, 110, Taiwan. E-mail: [email protected]
(Received 19 September 2013; accepted 13 November 2013) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2013.868603
Galectin-1 and nasopharyngeal carcinoma most common cancer in Taiwan, with an approximate incidence rate of 9.99 per 100 000 in males and 2.98 per 100 000 in females [9]. Thus, early diagnosis, exact histologic grading, and accurate prediction are critical for developing better treatment options and improving the prognosis of NPC. A future research goal might be based upon finding novel prognostic factors to precisely predict the biological behavior of the tumors. Hence, we conducted a cohort study to examine the effect of galectin-1 expression on the survival rate of patients with NPC. An examination of the amount of galectin-1 expression in NPC will, hopefully, provide useful information to physicians for selecting the optimal therapy, and to researchers working to identify the potentials of immunotherapy for patients with NPC. Material and methods Patients and tumor specimens The Chi Mei Medical Center institutional review board approved procurement of formalin-fixed NPC tissue for this study. Available paraffin-embedded tissue blocks were retrieved from 124 patients with NPC who had undergone biopsies between January 1998 and December 2002 during their initial diagnosis. None of the patients had distant metastasis at the time of diagnosis. Two pathologists reappraised the histological subtypes according to the current WHO classification. The tumor staging was reappraised using the 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/ AJCC) staging system [10]. Cell culture and immunoblotting analysis Dysplastic oral keratinocyte (DOK) and NPCderived HONE-1 and TW01 cell lines were maintained in Dulbecco’s modified Eagle’s medium/ Hams-F12 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; Biological Industry, Kibbutz Beit Haemek, Israel) in a 37 C, 5% CO2 incubator. Western blot assays were performed to evaluate the expression levels of galectin-1. Equal amounts of total protein (25 mg) extracted from the cell lines under various conditions were separated on 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel NuPAGE (Invitrogen), and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK). After they had been blocked with 5% skimmed milk in TBST (Tris-buffered saline and Tween 20) buffer at room temperature for 1 h, the membranes
537
were probed with antibodies at 48 C overnight against galectin-1 (1:1000) (cat. no. 437400; Invitrogen) and GADPH (glyceraldehyde-3-phosphate dehydrogenase) as a loading control (Clone 6C5, 1:10 000; Millipore, Beverly, MA, USA). After they had been incubated with the secondary antibody at room temperature for 1.5 h, protein expression was detected using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences). Immunohistochemical staining and assessment of galectin-1 expression Tissue sections 3 m thick were cut from paraffinembedded blocks. Slides were deparaffinized with xylene, rehydrated with ethanol, and heated in a microwave oven for 7 min to retrieve antigen epitopes in a 10 mM citrate buffer (pH 6). Endogenous peroxidase was quenched using 3% H2O2 treatment. Slides were washed with Tris buffered saline for 15 min, and then incubated with primary monoclonal antibodies targeting Gal-1 (1:200) (cat. no. 437400; Invitrogen) for 1 h. Primary antibodies were detected using a peroxidase-based kit (ChemMate EnVision kit K5007; DAKO, Carpinteria, CA, USA). The slides were incubated with secondary antibodies for 30 min, developed with 3,3-diaminobenzidine for 5 min, and then counterstained with hematoxylin. A mammary invasive ductal carcinoma cell line known to express galectin-1 was used as a positive control. Rabbit serum IgG was used to replace primary antibody as a negative control. Without prior knowledge of the patients’ tumor stages and the outcomes of their treatment, two pathologists (H.Y.H and C.F.L.) used a multi-headed microscope to score galectin-1 immunoexpression to arrive at a consensus. Galectin1 staining is interpreted using the immunohistochemistry H-score: H = (percentage score) (intensity score + 1). The percentage score ranged from 0 to 4, based on the proportion of nuclear-stained tumor cells (0, 50%). The intensity score ranged from 0 to 3 (0, negative staining; 1, weak nuclear staining; 2, moderate nuclear staining; and 3, strong nuclear staining), as described previously [11]. Treatment and follow-up All 124 patients were regularly monitored after radiotherapy until death or their last appointment. The mean follow-up duration was 67.0 months (range 3–141). Seven patients were not available for image studies after therapy, but the remaining 117 patients underwent a complete course of radiotherapy (total dose ‡ 7000 cGy) and also cisplatin-based chemotherapy if they reached
538
S.-L. Chang et al. DOK
stages II–IV of NPC, in accordance with the published protocol. The WHO reported a set of criteria that include completing clinical examinations and imaging modalities to assess treatment responses. Ninety-four patients met all the criteria, and seven patients met some of the same criteria.
GAPDH
Figure 1. Immunoblotting analyses showed that the galectin-1 protein level was not detected in dysplastic oral keratinocyte (DOK) cells. However, it was expressed in nasopharyngeal carcinoma (NPC)-derived HONE and TW01 cells.
Prognostic impact of galectin-1 expression In a mean follow-up of 67.0 months (range 3–141), local recurrence, distant metastasis, and tumorassociated mortality developed with a median duration of 21, 21, and 33 months, respectively (Table II). With respect to the three end point analyses, T3– 4 status, N2–3 status, and stages III and IV at diagnosis were all significant predictors of disease Table I. Associations between galectin-1 expression and other important clinicopathologic variables.
Results Galectin-1 was highly expressed in HONE-1 and TW01 cells Galectin-1 protein was not detected in DOK1 cells. However, it was highly expressed in HONE-1 and TW01 cells lines (Figure 1), which suggests that galectin-1 is involved in the carcinogenesis of NPC. Immunohistochemical expression of galectin-1 and its associations with clinicopathological variables The 124 patients with NPC (95 men, 29 women) had a mean age of 48.6 years (range 20–83) (Table I). Seven patients were classified as stage I, 31 as stage II, 46 as stage III, and 40 as stage IV. Five patients had keratinizing squamous cell carcinoma, 54 had differentiated non-keratinizing carcinoma, and 65 had undifferentiated non-keratinizing carcinoma. Although galectin-1 immunoexpression was rarely detected in the non-neoplastic nasopharyngeal mucosal epithelia, it was expressed in notable amounts in a certain number of cases of NPC with a cytoplasmic pattern (Figure 2). A higher level of galectin-1 expression was associated with a more advanced primary tumor classification (p = 0.009) and a more advanced AJCC stage (III–IV; p = 0.019).
TWO1
Galectin-1
Statistical analysis SPSS 14 for Windows was used for all statistical analyses. The associations of galectin-1 expression status with various clinicopathological parameters were evaluated using a 2 or Fisher’s exact test. The end point analyses were local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), and disease-specific survival (DSS), all of which were calculated from the starting date of radiotherapy to the date of death or the last appointment. Patients who stopped attending their follow-up appointments were censored on the latest follow-up date. Survival curves were plotted using the Kaplan–Meier method, and log-rank tests were done to evaluate prognostic differences between the groups. Multivariate analysis was done using the Cox proportional hazards model. For all analyses, two-sided tests of significance were used; significance was set at p < 0.05.
HONE
Galectin-1 expression Characteristic
Low
High
p value
Male
46
49
0.524
Female
16
13
