Pathology – Research and Practice 210 (2014) 649–655
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Original Article
Activated leukocyte cell adhesion molecule (ALCAM/CD166) expression in head and neck squamous cell carcinoma (HNSSC) Till Sebastian Clauditz a,1 , Kirsten von Rheinbaben a,1 , Patrick Lebok a , Sarah Minner a , Michael Tachezy e , Kerstin Borgmann d , Rainald Knecht b , Guido Sauter a , Waldemar Wilczak a , Marco Blessmann c,1 , Adrian Münscher b,∗,1 a
Institute of Pathology, University Medical Center Hamburg-Eppendorf, Germany Department of Otorhinolaryngology, University Medical Center Hamburg-Eppendorf, Germany c Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Germany d Laboratory of Radiobiology and Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Germany e Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Germany b
a r t i c l e
i n f o
Article history: Received 22 February 2014 Received in revised form 22 May 2014 Accepted 19 June 2014 Keywords: Head and neck carcinomas Cell adhesion Prognostic marker CD166 ALCAM
a b s t r a c t Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed in a number of malignancies (e.g. prostate, breast, squamous cell carcinoma of the esophagus, lung and head and neck tumors). Based on studies in which ALCAM showed prognostic relevance in several carcinomas, it has been discussed as a potential therapeutic target. We evaluate its expression in head and neck squamous cell carcinomas (HNSCCs). A tissue microarray was constructed from more than 400 HNSCCs. Slides were analyzed by immunohistochemistry for ALCAM. Membranous and cytoplasmic ALCAM positivity were rated separately. The tumors were combined into (a) cases with membranous staining and (b) cases with cytoplasmic staining, independently from membranous/cytoplasmic co-expression. We found staining in 70.3% of interpretable HNSCCs. Pure membranous staining was found in 12.4% of tumors, with cytoplasmic positivity in 40.1% of cases, and membranous/cytoplasmic co-expression in 17.9%. No significant association between ALCAM positivity and clinical parameters was found. No significant association between ALCAM expression and survival data was observed for all tumors. The frequent expression of ALCAM (70.3%) in head and neck squamous cell carcinomas does not support an important role for HNSCC biology. The increased levels of ALCAM suggest the existence of a therapeutic window for potential anti-ALCAM therapies. © 2014 Elsevier GmbH. All rights reserved.
Introduction Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the cell surface immunoglobulin superfamily. It is involved in multiple processes including embryogenesis, hematopoiesis, angiogenesis, and immune response and can be found in several organs and malignancies [1–4].
∗ Corresponding author at: Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel.: +49 40 7410 0047; fax: +49 40 7410 56319. E-mail addresses: [email protected], [email protected] (A. Münscher). 1 Equal contribution. http://dx.doi.org/10.1016/j.prp.2014.06.012 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
Previous studies show variable ALCAM expression in malignancies. Increased (membranous and/or cytoplasmic) ALCAM expression was found in several carcinoma entities, including pancreas, colorectal, prostate, ovary, esophagus and oral squamous cell carcinoma and malignant melanoma [5–10]. For example, colorectal cancer and esophageal squamous cell carcinoma showed a significant correlation between ALCAM immunohistochemical staining and tumor stage, lymph node status and clinical stage [6,7]. However, downregulated ALCAM expression was found in highgrade prostate cancer as well as breast cancer and was reported to be associated with unfavorable tumor phenotypes and prognosis [4,11,12]. For oral squamous cell carcinoma the findings are ambiguous. Sawhney et al. showed a significant association between positive cytoplasmic ALCAM expression and clinical stage, tumor size and disease-free survival, whereas van den Brandt and colleagues
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reported an association between membranous ALCAM expression and nodal metastasis [13,14]. Based on the results of previous studies on oral squamous cell cancer, the aim of this study was to analyze membranous as well as cytoplasmic ALCAM expression in the whole head and neck region, including squamous cell carcinomas of the oral cavity, hypo-/pharynx and larynx. Materials and methods Specimen collection and TMA construction The tissue microarray was constructed from a total of 453 HNSCC specimens from formalin-fixed, paraffin-embedded archived tissue samples of the Institute of Pathology at the University Medical Center Hamburg-Eppendorf, as described previously [15]. One tissue core of 0.6 mm was punched out of each case and transferred in a TMA format as described previously [16]. The local ethics committee approved the usage of tissue microarrays and tumor specimens for research purposes. Only surgical specimens of tumor ectomy without previous therapy were used for tissue microarray construction and were reviewed by one pathologist (TSC). Twenty control tissue samples from different head and neck regions (oral cavity, oropharynx, hypopharynx and larynx) of non-tumor patients were punched on the same TMA to evaluate ALCAM expression in normal squamous tissue. The pathologic stages (pT, pN) and tumor grade were obtained from the primary report of the Institute of Pathology. Clinical data (gender, age, cM, time to recurrence and overall survival) were obtained from the medical report. Survival data were available from 441 patients. The mean follow-up period was 41 months. Recurrence was defined as tumor relapse after operation with or without adjuvant therapy. An overview of clinical and pathological data is shown in Table 1. Consecutive, freshly cut sections of the tissue microarrays were used for immunohistochemical analysis and H&E stained reference. Immunohistochemistry Freshly cut TMA sections were analyzed on one day in a single experiment. ALCAM (CD166) expression was detected using a well-established [9,10] monoclonal antibody (clone: MOG/07; Novocastra® ) after peroxidase blocking with H2 O2 (DAKO S2023) for 10 min. High-temperature pretreatment of slides was conducted in an autoclave in citrate buffer, pH 7.8 for 5 min. The Envision system (DAKO 5007) was used to visualize the immunostaining. ALCAM staining was evaluated separately by two experienced pathologists (TSC & PL) using an established scoring system being described in several previous studies [9,10]. In this scoring system, the staining intensity (0, 1+, 2+, 3+) and the fraction of positive tumor cells were scored for each tissue spot, and a final IHC score (negative, weak, moderate strong) is built from these parameters as described before [9,10]. In case of discrepancies, tissue spots were re-analyzed by both pathologists and a consensus result was assigned. In addition, the localization of the staining was noted (membranous only, cytoplasmic only, membranous and cytoplasmic co-staining). For subset analyses, tumors showing any ALCAM expression (irrespective of the score or localization) were combined into one group of “ALCAM positive” cancers. Statistical analysis The statistical analysis was conducted using JMP 10.0 software (SAS institute Inc., Cary, North Carolina, USA). All p-values were two-sided and p-values 0.5) or cytoplasmic expression (Fig. 2c and d; p > 0.3) was analysed. In addition, there was also no impact of the staining localization (Fig. 2e and f; p > 0.3), or if tumors with positive staining were combined into one group (Fig. 2g and h; p > 0.8). There was also no association with patient prognosis if tumors of the oral cavity (Fig. 3a and b; p > 0.3) oropharynx (Fig. 3c and d; p > 0.3), or larynx (Fig. 3e and f; p > 0.8) were analysed separately. Supplementary Fig. 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2014.06.012.
Discussion The results of our study show that ALCAM is frequently expressed in the cytoplasm and/or at the membrane in head and neck squamous cell carcinomas, but argue against a relevant role of ALCAM expression for HNSCC aggressiveness.
Fig. 1. Immunohistochemical ALCAM expression in HNSCC and normal squamous epithelium: (A–C) HNSCC showing weak, moderate and strong cytoplasmic ALCAM expression; inlet C: representative negative immunohistochemistry. (D–F) HNSCC showing weak, moderate and strong membranous ALCAM expression.
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Table 3 CD166 (ALCAM) accumulation regarding clinicopathologic characteristics. HNSCC CD166
Sex Male Female Age (years) 70 pT-category T1 T2 T3 T4 pN-category N0 N1 N2 N3 cM-category M0 M1 Grade I II III
Oral cavity CD166
Neg. n (%)
Pos. n (%)
78 (30.1) 25 (28.7)
181 (69.9) 62 (71.3)
3 (21.4) 15 (30) 35 (31.3) 24 (22.6) 26 (40.6)
11 (78.6) 35 (70) 77 (68.7) 82 (77.4) 38 (59.4)
23 (32.9) 28 (27.2) 18 (29.5) 34 (31.5)
47 (67.1) 75 (72.8) 43 (70.5) 74 (68.5)
44 (27.3) 25 (44.6) 28 (27.2) 6 (27.3)
117 (72.7) 31 (55.4) 75 (72.8) 16 (72.7)
97 (30.8) 6 (27.3)
218 (69.2) 16 (72.7)
6 (25) 74 (30.8) 23 (29.5)
18 (75) 166 (69.2) 55 (70.5)
II III IV
Neg. n (%)
Pos. n (%)
35 (33) 13 (29.6)
71 (67) 31 (70.4)
2 (25) 5 (27.8) 17 (37) 9 (21.9) 15 (40.5)
6 (75) 13 (72.2) 29 (63) 32 (78.1) 22 (59.5)
11 (31.4) 13 (25) 8 (42.1) 16 (36.4)
24 (68.6) 39 (75) 11 (57.9) 28 (63.6)
17 (22.7) 15 (57.7) 13 (31.7) 3 (37.5)
58 (77.3) 11 (42.3) 28 (68.3) 5 (62.5)
44 (31.7) 4 (40)
95 (68.3) 6 (60)
5 (45.5) 35 (32.7) 8 (25)
6 (54.5) 72 (67.3) 24 (75)
0.807
Pos. n (%)
23 (37.7) 8 (34.8)
38 (62.3) 15 (65.2)
0 7 (50) 10 (30.3) 9 (36) 5 (45.5)
1 (100) 7 (50) 23 (69.7) 16 (64) 6 (54.5)
6 (35.3) 10 (40) 7 (41.2) 8 (32)
11 (64.7) 15 (60) 10 (58.8) 17 (68)
10 (41.7) 5 (41.7) 14 (35) 2 (25)
14 (58.3) 7 (58.3) 26 (65) 6 (75)
31 (39.7) 0
47 (60.3) 3 (100)
1 (50) 23 (37.7) 7 (33.3)
1 (50) 38 (62.3) 14 (66.7)
18 (78.3) 22 (88) 14 (46.7) 44 (65.7)
Positive cytoplasmic and/or membranous staining was found in 70.32% of HNSCC in our study. This fraction is comparable with results from earlier studies reporting between 61 and 67% [13,14] in cancers of the oral cavity. In contrast to these earlier studies employing large section analysis, we measured ALCAM levels in a TMA containing only one 0.6 mm TMA spot per case. The almost identical fraction of positive cases found in large sections and in TMA spots provides another example that results obtained from TMA spots are typically representative of results achieved by large section analysis [17–21], and also agues against major intratumoral heterogeneity of ALCAM expression in HNSCC.
72 (78.3) 16 (80)
1 (20) 3 (16.7) 8.24.24
4 (80) 15 (83.3) 25.75.76
6 (15) 6 (37.5)
34 (85) 10 (62.5)
6 (33.3) 5 (19.2) 3 (12) 10 (25.6)
12 (66.7) 21 (80.8) 22 (88) 29 (74.4)
17 (27.4) 5 (27.8) 1 (4.6) 1 (16.7)
45 (72.6) 13 (72.2) 21 (95.5) 5 (83.3)
22 (22.5) 2 (22.2)
76 (77.5) 7 (77.8)
0
11 (100) 56 (77.8) 17 (68)
0.459
0.445
0.079
0.766
0.871
0.034
16 (22.2) 8 (32) 0.427
4 (66.7) 4 (40) 7 (38.9) 16 (32)
2 (33.3) 6 (60) 11 (61.1) 34 (68)
p-Value 0.862
0.085
0.0056 5 (21.7) 3 (12) 16 (53.3) 23 (34.3)
20 (21.7) 4 (20)
0.819
0.440
0.229
Pos. n (%)
0.915
0.592
0.826
Neg. n (%)
0.575
0.0144
0.726
p-Value 0.804
0.483
0.096
34 (70.8) 45 (77.6) 40 (60.6) 115 (70.1)
Neg. n (%)
0.396
0.854
14 (29.7) 13 (22.4) 26 (39.4) 49 (29.9)
p-Value
Hypopharynx/larynx CD166
0.677
0.151
UICC TNM stage I
p-Value
Oropharynx CD166
0.865 5 (26.3) 6 (26.1) 3 (16.7) 10 (21.3)
14 (73.7) 17 (73.9) 15 (83.3) 37 (78.7)
The higher fraction of ALCAM positivity in cancers as compared to normal squamous tissues suggests that ALCAM is up-regulated during cancer development. The striking lack of associations with tumor phenotype or patient prognosis – irrespectively of the staining levels or subcellular localization – strongly argues against a relevant role of ALCAM expression for HNSCC progression. Our results are in contrast to two earlier studies analyzing ALCAM in cancers of the oral cavity. Sawhney et al. suggested a role of cytoplasmic ALCAM overexpression for shortened recurrence-free survival in a cohort of 107 OSCCs [13], and van den Brand et al. reported a link between membranous ALCAM positivity and presence of lymph
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Fig. 2. Recurrent-free and overall survival curves of analysed HNSCCs regarding the locus of expression, (A+B) membranous and (C+D) cytoplasmic, the expression pattern (E+F), as well as the pure positivity (G+H).
node metastases in a small cohort of 41 cancers [14]. It is possible, that the small numbers of samples included in these studies have led to occasional significant results that do not hold true in larger cohorts like ours. However, published findings on the prognostic value of ALCAM demonstrate that ALCAM may be linked to either good or poor prognosis depending on the cancer type. For example, other and us reported an association between increased ALCAM
expression and low malignant phenotype in prostate and breast cancer [4,11,12], whereas ALCAM expression (particularly if cytoplasmic) has been linked to adverse tumor features in cancers of the digestic system rather [22]. Our present study does not support a clinical value of ALCAM testing in the different types of HNSCC. ALCAM expression was often both cytoplasmic and membranous in our study, but there were also tumors with predominant
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Fig. 3. Recurrent-free and overall survival curves regarding the HNSCC subgroups, OSCC (A+B), oropharynx (C+D) and larynx (E+F).
staining either at the membrane or in the cytoplasm. However, we did not find specific associations between HNSCC features and the subcellular localization of ALCAM in our study (Fig. 2). Previous studies have suggested that the subcellular localization of ALCAM is related to the activity of specific cellular functions [8,23]. For example, studies on ovary carcinoma cells showed internalization of ALCAM to the cytoplasmic level [24]. Additionally, Nelissen et al. [24] showed that the lateral mobility of ALCAM was restrained by the actin cytoskeleton. This may be evidence of cell-dependent rearrangement and/or a reason for the sustained invasive or metastatic tumor growth, and supports the hypothesis that cytoplasmic ALCAM accumulation might be a result of structural changes in the protein. Increased cytoplasmic ALCAM expression was also found to be associated with a worse outcome in other studies on ovarian and breast carcinomas [8,23]. Mezzanzanica et al. [8] analyzed the subcellular localization of ALCAM in ovarian carcinomas and found predominantly cytoplasmic expression. They assumed that re-location of membrane-bound ALCAM to the cytoplasm led to diminished cell-to-cell adhesion and therefore
promoted cell migration. Other authors found that increased membranous ALCAM in different malignancies (such as colon carcinoma and malignant melanoma) was associated with poor prognosis and progression [7,25]. The influence of high membranous ALCAM expression on the metastatic growth of melanoma cells was shown in mice in which the experimental group inoculated with melanoma cells co-expressing wild-type ALCAM and a transmembrane, amino-terminally truncated ALCAM construct developed more metastatic lesions than the control group inoculated with melanoma cells expressing only endogenous wild-type ALCAM [26]. It was also shown that Tiam1 molecule (T-lymphoma invasion and metastasis 1) can regulate ALCAM localization on the cell membrane and therefore may affect the invasive growth of metastatic melanoma cells [27]. Despite a clear-cut relevance of the subcellular localization of ALCAM for tumor phenotype or aggressiveness, proper determination of the intracellular localization may become relevant if ALCAM is regarded as a therapy target [28]. It has been suggested that membranous ALCAM, which is permanently internalized by the
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endocytic clathrin-dependent pathway [28], may be used as a shuttle for anti-ALCAM drugs that may not be able to enter the cell on its own, i.e. therapeutic antibodies [29]. In addition to its potential as a therapeutic target, ALCAM may also have clinical relevance regarding the therapeutic response. Hong et al. [30] reported that ALCAM-silenced pancreatic carcinoma cells showed resistance against gemcitabine and actinomycin-D. For breast cancer patients, Ihnen et al. reported that tumors with high ALCAM mRNA levels had a significantly longer overall survival upon adjuvant chemotherapy compared to those with low mRNA levels and concluded that ALCAM expression in breast carcinomas might be a suitable predictive factor for responsiveness in adjuvant chemotherapy settings [31]. In conclusion, the results of our study do not support an important role of ALCAM expression for HNSCC biology. The increased levels of ALCAM in cancers as compared to normal tissues suggest the existence of a therapeutic window for potential anti-ALCAM therapies. References [1] C. Fournier-Thibault, O. Pourquie, T. Rouaud, N.M. Le Douarin, BEN/SC1/DMGRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation, J. Neurosci. 19 (1999) 1382–1392. [2] O. Ohneda, K. Ohneda, F. Arai, J. Lee, T. Miyamoto, Y. Fukushima, D. Dowbenko, L.A. Lasky, T. Suda, ALCAM (CD166): its role in hematopoietic and endothelial development, Blood 98 (2001) 2134–2142. [3] G.W. Swart, Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration, Eur. J. Cell Biol. 81 (2002) 313–321. [4] S.F. Ofori-Acquah, J.A. King, Activated leukocyte cell adhesion molecule: a new paradox in cancer, Transl. Res. 151 (2008) 122–128. [5] L.C. van Kempen, J.M. Nelissen, W.G. Degen, R. Torensma, U.H. Weidle, H.P. Bloemers, C.G. Figdor, G.W. Swart, Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)–ALCAM interaction, J. Biol. Chem. 276 (2001) 25783–25790. [6] A. Verma, N.K. Shukla, S.V. Deo, S.D. Gupta, R. Ralhan, MEMD/ALCAM: a potential marker for tumor invasion and nodal metastasis in esophageal squamous cell carcinoma, Oncology 68 (2005) 462–470. [7] W. Weichert, T. Knosel, J. Bellach, M. Dietel, G. Kristiansen, ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival, J. Clin. Pathol. 57 (2004) 1160–1164. [8] D. Mezzanzanica, M. Fabbi, M. Bagnoli, S. Staurengo, M. Losa, E. Balladore, P. Alberti, L. Lusa, A. Ditto, S. Ferrini, M.A. Pierotti, M. Barbareschi, S. Pilotti, S. Canevari, Subcellular localization of activated leukocyte cell adhesion molecule is a molecular predictor of survival in ovarian carcinoma patients, Clin. Cancer Res. 14 (2008) 1726–1733. [9] S. Minner, F. Kraetzig, M. Tachezy, E. Kilic, M. Graefen, W. Wilczak, C. Bokemeyer, H. Huland, G. Sauter, T. Schlomm, Low activated leukocyte cell adhesion molecule expression is associated with advanced tumor stage and early prostate-specific antigen relapse in prostate cancer, Hum. Pathol. 42 (2011) 1946–1952. [10] M. Tachezy, H. Zander, A.H. Marx, F. Gebauer, T. Rawnaq, J.T. Kaifi, G. Sauter, J.R. Izbicki, M. Bockhorn, ALCAM (CD166) expression as novel prognostic biomarker for pancreatic neuroendocrine tumor patients, J. Surg. Res. 170 (2011) 226–232. [11] G. Kristiansen, C. Pilarsky, C. Wissmann, C. Stephan, L. Weissbach, V. Loy, S. Loening, M. Dietel, A. Rosenthal, ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions, Prostate 54 (2003) 34–43. [12] J.A. King, S.F. Ofori-Acquah, T. Stevens, A.B. Al-Mehdi, O. Fodstad, W.G. Jiang, Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator, Breast Cancer Res. 6 (2004) R478–R487.
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Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Activated leukocyte cell adhesion molecule (ALCAM/CD166) expression in head and neck squamous cell carcinoma (HNSSC) Till Sebastian Clauditz a,1 , Kirsten von Rheinbaben a,1 , Patrick Lebok a , Sarah Minner a , Michael Tachezy e , Kerstin Borgmann d , Rainald Knecht b , Guido Sauter a , Waldemar Wilczak a , Marco Blessmann c,1 , Adrian Münscher b,∗,1 a
Institute of Pathology, University Medical Center Hamburg-Eppendorf, Germany Department of Otorhinolaryngology, University Medical Center Hamburg-Eppendorf, Germany c Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Germany d Laboratory of Radiobiology and Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Germany e Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Germany b
a r t i c l e
i n f o
Article history: Received 22 February 2014 Received in revised form 22 May 2014 Accepted 19 June 2014 Keywords: Head and neck carcinomas Cell adhesion Prognostic marker CD166 ALCAM
a b s t r a c t Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed in a number of malignancies (e.g. prostate, breast, squamous cell carcinoma of the esophagus, lung and head and neck tumors). Based on studies in which ALCAM showed prognostic relevance in several carcinomas, it has been discussed as a potential therapeutic target. We evaluate its expression in head and neck squamous cell carcinomas (HNSCCs). A tissue microarray was constructed from more than 400 HNSCCs. Slides were analyzed by immunohistochemistry for ALCAM. Membranous and cytoplasmic ALCAM positivity were rated separately. The tumors were combined into (a) cases with membranous staining and (b) cases with cytoplasmic staining, independently from membranous/cytoplasmic co-expression. We found staining in 70.3% of interpretable HNSCCs. Pure membranous staining was found in 12.4% of tumors, with cytoplasmic positivity in 40.1% of cases, and membranous/cytoplasmic co-expression in 17.9%. No significant association between ALCAM positivity and clinical parameters was found. No significant association between ALCAM expression and survival data was observed for all tumors. The frequent expression of ALCAM (70.3%) in head and neck squamous cell carcinomas does not support an important role for HNSCC biology. The increased levels of ALCAM suggest the existence of a therapeutic window for potential anti-ALCAM therapies. © 2014 Elsevier GmbH. All rights reserved.
Introduction Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the cell surface immunoglobulin superfamily. It is involved in multiple processes including embryogenesis, hematopoiesis, angiogenesis, and immune response and can be found in several organs and malignancies [1–4].
∗ Corresponding author at: Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel.: +49 40 7410 0047; fax: +49 40 7410 56319. E-mail addresses: [email protected], [email protected] (A. Münscher). 1 Equal contribution. http://dx.doi.org/10.1016/j.prp.2014.06.012 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
Previous studies show variable ALCAM expression in malignancies. Increased (membranous and/or cytoplasmic) ALCAM expression was found in several carcinoma entities, including pancreas, colorectal, prostate, ovary, esophagus and oral squamous cell carcinoma and malignant melanoma [5–10]. For example, colorectal cancer and esophageal squamous cell carcinoma showed a significant correlation between ALCAM immunohistochemical staining and tumor stage, lymph node status and clinical stage [6,7]. However, downregulated ALCAM expression was found in highgrade prostate cancer as well as breast cancer and was reported to be associated with unfavorable tumor phenotypes and prognosis [4,11,12]. For oral squamous cell carcinoma the findings are ambiguous. Sawhney et al. showed a significant association between positive cytoplasmic ALCAM expression and clinical stage, tumor size and disease-free survival, whereas van den Brandt and colleagues
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reported an association between membranous ALCAM expression and nodal metastasis [13,14]. Based on the results of previous studies on oral squamous cell cancer, the aim of this study was to analyze membranous as well as cytoplasmic ALCAM expression in the whole head and neck region, including squamous cell carcinomas of the oral cavity, hypo-/pharynx and larynx. Materials and methods Specimen collection and TMA construction The tissue microarray was constructed from a total of 453 HNSCC specimens from formalin-fixed, paraffin-embedded archived tissue samples of the Institute of Pathology at the University Medical Center Hamburg-Eppendorf, as described previously [15]. One tissue core of 0.6 mm was punched out of each case and transferred in a TMA format as described previously [16]. The local ethics committee approved the usage of tissue microarrays and tumor specimens for research purposes. Only surgical specimens of tumor ectomy without previous therapy were used for tissue microarray construction and were reviewed by one pathologist (TSC). Twenty control tissue samples from different head and neck regions (oral cavity, oropharynx, hypopharynx and larynx) of non-tumor patients were punched on the same TMA to evaluate ALCAM expression in normal squamous tissue. The pathologic stages (pT, pN) and tumor grade were obtained from the primary report of the Institute of Pathology. Clinical data (gender, age, cM, time to recurrence and overall survival) were obtained from the medical report. Survival data were available from 441 patients. The mean follow-up period was 41 months. Recurrence was defined as tumor relapse after operation with or without adjuvant therapy. An overview of clinical and pathological data is shown in Table 1. Consecutive, freshly cut sections of the tissue microarrays were used for immunohistochemical analysis and H&E stained reference. Immunohistochemistry Freshly cut TMA sections were analyzed on one day in a single experiment. ALCAM (CD166) expression was detected using a well-established [9,10] monoclonal antibody (clone: MOG/07; Novocastra® ) after peroxidase blocking with H2 O2 (DAKO S2023) for 10 min. High-temperature pretreatment of slides was conducted in an autoclave in citrate buffer, pH 7.8 for 5 min. The Envision system (DAKO 5007) was used to visualize the immunostaining. ALCAM staining was evaluated separately by two experienced pathologists (TSC & PL) using an established scoring system being described in several previous studies [9,10]. In this scoring system, the staining intensity (0, 1+, 2+, 3+) and the fraction of positive tumor cells were scored for each tissue spot, and a final IHC score (negative, weak, moderate strong) is built from these parameters as described before [9,10]. In case of discrepancies, tissue spots were re-analyzed by both pathologists and a consensus result was assigned. In addition, the localization of the staining was noted (membranous only, cytoplasmic only, membranous and cytoplasmic co-staining). For subset analyses, tumors showing any ALCAM expression (irrespective of the score or localization) were combined into one group of “ALCAM positive” cancers. Statistical analysis The statistical analysis was conducted using JMP 10.0 software (SAS institute Inc., Cary, North Carolina, USA). All p-values were two-sided and p-values 0.5) or cytoplasmic expression (Fig. 2c and d; p > 0.3) was analysed. In addition, there was also no impact of the staining localization (Fig. 2e and f; p > 0.3), or if tumors with positive staining were combined into one group (Fig. 2g and h; p > 0.8). There was also no association with patient prognosis if tumors of the oral cavity (Fig. 3a and b; p > 0.3) oropharynx (Fig. 3c and d; p > 0.3), or larynx (Fig. 3e and f; p > 0.8) were analysed separately. Supplementary Fig. 1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2014.06.012.
Discussion The results of our study show that ALCAM is frequently expressed in the cytoplasm and/or at the membrane in head and neck squamous cell carcinomas, but argue against a relevant role of ALCAM expression for HNSCC aggressiveness.
Fig. 1. Immunohistochemical ALCAM expression in HNSCC and normal squamous epithelium: (A–C) HNSCC showing weak, moderate and strong cytoplasmic ALCAM expression; inlet C: representative negative immunohistochemistry. (D–F) HNSCC showing weak, moderate and strong membranous ALCAM expression.
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Table 3 CD166 (ALCAM) accumulation regarding clinicopathologic characteristics. HNSCC CD166
Sex Male Female Age (years) 70 pT-category T1 T2 T3 T4 pN-category N0 N1 N2 N3 cM-category M0 M1 Grade I II III
Oral cavity CD166
Neg. n (%)
Pos. n (%)
78 (30.1) 25 (28.7)
181 (69.9) 62 (71.3)
3 (21.4) 15 (30) 35 (31.3) 24 (22.6) 26 (40.6)
11 (78.6) 35 (70) 77 (68.7) 82 (77.4) 38 (59.4)
23 (32.9) 28 (27.2) 18 (29.5) 34 (31.5)
47 (67.1) 75 (72.8) 43 (70.5) 74 (68.5)
44 (27.3) 25 (44.6) 28 (27.2) 6 (27.3)
117 (72.7) 31 (55.4) 75 (72.8) 16 (72.7)
97 (30.8) 6 (27.3)
218 (69.2) 16 (72.7)
6 (25) 74 (30.8) 23 (29.5)
18 (75) 166 (69.2) 55 (70.5)
II III IV
Neg. n (%)
Pos. n (%)
35 (33) 13 (29.6)
71 (67) 31 (70.4)
2 (25) 5 (27.8) 17 (37) 9 (21.9) 15 (40.5)
6 (75) 13 (72.2) 29 (63) 32 (78.1) 22 (59.5)
11 (31.4) 13 (25) 8 (42.1) 16 (36.4)
24 (68.6) 39 (75) 11 (57.9) 28 (63.6)
17 (22.7) 15 (57.7) 13 (31.7) 3 (37.5)
58 (77.3) 11 (42.3) 28 (68.3) 5 (62.5)
44 (31.7) 4 (40)
95 (68.3) 6 (60)
5 (45.5) 35 (32.7) 8 (25)
6 (54.5) 72 (67.3) 24 (75)
0.807
Pos. n (%)
23 (37.7) 8 (34.8)
38 (62.3) 15 (65.2)
0 7 (50) 10 (30.3) 9 (36) 5 (45.5)
1 (100) 7 (50) 23 (69.7) 16 (64) 6 (54.5)
6 (35.3) 10 (40) 7 (41.2) 8 (32)
11 (64.7) 15 (60) 10 (58.8) 17 (68)
10 (41.7) 5 (41.7) 14 (35) 2 (25)
14 (58.3) 7 (58.3) 26 (65) 6 (75)
31 (39.7) 0
47 (60.3) 3 (100)
1 (50) 23 (37.7) 7 (33.3)
1 (50) 38 (62.3) 14 (66.7)
18 (78.3) 22 (88) 14 (46.7) 44 (65.7)
Positive cytoplasmic and/or membranous staining was found in 70.32% of HNSCC in our study. This fraction is comparable with results from earlier studies reporting between 61 and 67% [13,14] in cancers of the oral cavity. In contrast to these earlier studies employing large section analysis, we measured ALCAM levels in a TMA containing only one 0.6 mm TMA spot per case. The almost identical fraction of positive cases found in large sections and in TMA spots provides another example that results obtained from TMA spots are typically representative of results achieved by large section analysis [17–21], and also agues against major intratumoral heterogeneity of ALCAM expression in HNSCC.
72 (78.3) 16 (80)
1 (20) 3 (16.7) 8.24.24
4 (80) 15 (83.3) 25.75.76
6 (15) 6 (37.5)
34 (85) 10 (62.5)
6 (33.3) 5 (19.2) 3 (12) 10 (25.6)
12 (66.7) 21 (80.8) 22 (88) 29 (74.4)
17 (27.4) 5 (27.8) 1 (4.6) 1 (16.7)
45 (72.6) 13 (72.2) 21 (95.5) 5 (83.3)
22 (22.5) 2 (22.2)
76 (77.5) 7 (77.8)
0
11 (100) 56 (77.8) 17 (68)
0.459
0.445
0.079
0.766
0.871
0.034
16 (22.2) 8 (32) 0.427
4 (66.7) 4 (40) 7 (38.9) 16 (32)
2 (33.3) 6 (60) 11 (61.1) 34 (68)
p-Value 0.862
0.085
0.0056 5 (21.7) 3 (12) 16 (53.3) 23 (34.3)
20 (21.7) 4 (20)
0.819
0.440
0.229
Pos. n (%)
0.915
0.592
0.826
Neg. n (%)
0.575
0.0144
0.726
p-Value 0.804
0.483
0.096
34 (70.8) 45 (77.6) 40 (60.6) 115 (70.1)
Neg. n (%)
0.396
0.854
14 (29.7) 13 (22.4) 26 (39.4) 49 (29.9)
p-Value
Hypopharynx/larynx CD166
0.677
0.151
UICC TNM stage I
p-Value
Oropharynx CD166
0.865 5 (26.3) 6 (26.1) 3 (16.7) 10 (21.3)
14 (73.7) 17 (73.9) 15 (83.3) 37 (78.7)
The higher fraction of ALCAM positivity in cancers as compared to normal squamous tissues suggests that ALCAM is up-regulated during cancer development. The striking lack of associations with tumor phenotype or patient prognosis – irrespectively of the staining levels or subcellular localization – strongly argues against a relevant role of ALCAM expression for HNSCC progression. Our results are in contrast to two earlier studies analyzing ALCAM in cancers of the oral cavity. Sawhney et al. suggested a role of cytoplasmic ALCAM overexpression for shortened recurrence-free survival in a cohort of 107 OSCCs [13], and van den Brand et al. reported a link between membranous ALCAM positivity and presence of lymph
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Fig. 2. Recurrent-free and overall survival curves of analysed HNSCCs regarding the locus of expression, (A+B) membranous and (C+D) cytoplasmic, the expression pattern (E+F), as well as the pure positivity (G+H).
node metastases in a small cohort of 41 cancers [14]. It is possible, that the small numbers of samples included in these studies have led to occasional significant results that do not hold true in larger cohorts like ours. However, published findings on the prognostic value of ALCAM demonstrate that ALCAM may be linked to either good or poor prognosis depending on the cancer type. For example, other and us reported an association between increased ALCAM
expression and low malignant phenotype in prostate and breast cancer [4,11,12], whereas ALCAM expression (particularly if cytoplasmic) has been linked to adverse tumor features in cancers of the digestic system rather [22]. Our present study does not support a clinical value of ALCAM testing in the different types of HNSCC. ALCAM expression was often both cytoplasmic and membranous in our study, but there were also tumors with predominant
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Fig. 3. Recurrent-free and overall survival curves regarding the HNSCC subgroups, OSCC (A+B), oropharynx (C+D) and larynx (E+F).
staining either at the membrane or in the cytoplasm. However, we did not find specific associations between HNSCC features and the subcellular localization of ALCAM in our study (Fig. 2). Previous studies have suggested that the subcellular localization of ALCAM is related to the activity of specific cellular functions [8,23]. For example, studies on ovary carcinoma cells showed internalization of ALCAM to the cytoplasmic level [24]. Additionally, Nelissen et al. [24] showed that the lateral mobility of ALCAM was restrained by the actin cytoskeleton. This may be evidence of cell-dependent rearrangement and/or a reason for the sustained invasive or metastatic tumor growth, and supports the hypothesis that cytoplasmic ALCAM accumulation might be a result of structural changes in the protein. Increased cytoplasmic ALCAM expression was also found to be associated with a worse outcome in other studies on ovarian and breast carcinomas [8,23]. Mezzanzanica et al. [8] analyzed the subcellular localization of ALCAM in ovarian carcinomas and found predominantly cytoplasmic expression. They assumed that re-location of membrane-bound ALCAM to the cytoplasm led to diminished cell-to-cell adhesion and therefore
promoted cell migration. Other authors found that increased membranous ALCAM in different malignancies (such as colon carcinoma and malignant melanoma) was associated with poor prognosis and progression [7,25]. The influence of high membranous ALCAM expression on the metastatic growth of melanoma cells was shown in mice in which the experimental group inoculated with melanoma cells co-expressing wild-type ALCAM and a transmembrane, amino-terminally truncated ALCAM construct developed more metastatic lesions than the control group inoculated with melanoma cells expressing only endogenous wild-type ALCAM [26]. It was also shown that Tiam1 molecule (T-lymphoma invasion and metastasis 1) can regulate ALCAM localization on the cell membrane and therefore may affect the invasive growth of metastatic melanoma cells [27]. Despite a clear-cut relevance of the subcellular localization of ALCAM for tumor phenotype or aggressiveness, proper determination of the intracellular localization may become relevant if ALCAM is regarded as a therapy target [28]. It has been suggested that membranous ALCAM, which is permanently internalized by the
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endocytic clathrin-dependent pathway [28], may be used as a shuttle for anti-ALCAM drugs that may not be able to enter the cell on its own, i.e. therapeutic antibodies [29]. In addition to its potential as a therapeutic target, ALCAM may also have clinical relevance regarding the therapeutic response. Hong et al. [30] reported that ALCAM-silenced pancreatic carcinoma cells showed resistance against gemcitabine and actinomycin-D. For breast cancer patients, Ihnen et al. reported that tumors with high ALCAM mRNA levels had a significantly longer overall survival upon adjuvant chemotherapy compared to those with low mRNA levels and concluded that ALCAM expression in breast carcinomas might be a suitable predictive factor for responsiveness in adjuvant chemotherapy settings [31]. In conclusion, the results of our study do not support an important role of ALCAM expression for HNSCC biology. The increased levels of ALCAM in cancers as compared to normal tissues suggest the existence of a therapeutic window for potential anti-ALCAM therapies. References [1] C. Fournier-Thibault, O. Pourquie, T. Rouaud, N.M. Le Douarin, BEN/SC1/DMGRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation, J. Neurosci. 19 (1999) 1382–1392. [2] O. Ohneda, K. Ohneda, F. Arai, J. Lee, T. Miyamoto, Y. Fukushima, D. Dowbenko, L.A. Lasky, T. Suda, ALCAM (CD166): its role in hematopoietic and endothelial development, Blood 98 (2001) 2134–2142. [3] G.W. Swart, Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration, Eur. J. Cell Biol. 81 (2002) 313–321. [4] S.F. Ofori-Acquah, J.A. King, Activated leukocyte cell adhesion molecule: a new paradox in cancer, Transl. Res. 151 (2008) 122–128. [5] L.C. van Kempen, J.M. Nelissen, W.G. Degen, R. Torensma, U.H. Weidle, H.P. Bloemers, C.G. Figdor, G.W. Swart, Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)–ALCAM interaction, J. Biol. Chem. 276 (2001) 25783–25790. [6] A. Verma, N.K. Shukla, S.V. Deo, S.D. Gupta, R. Ralhan, MEMD/ALCAM: a potential marker for tumor invasion and nodal metastasis in esophageal squamous cell carcinoma, Oncology 68 (2005) 462–470. [7] W. Weichert, T. Knosel, J. Bellach, M. Dietel, G. Kristiansen, ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival, J. Clin. Pathol. 57 (2004) 1160–1164. [8] D. Mezzanzanica, M. Fabbi, M. Bagnoli, S. Staurengo, M. Losa, E. Balladore, P. Alberti, L. Lusa, A. Ditto, S. Ferrini, M.A. Pierotti, M. Barbareschi, S. Pilotti, S. Canevari, Subcellular localization of activated leukocyte cell adhesion molecule is a molecular predictor of survival in ovarian carcinoma patients, Clin. Cancer Res. 14 (2008) 1726–1733. [9] S. Minner, F. Kraetzig, M. Tachezy, E. Kilic, M. Graefen, W. Wilczak, C. Bokemeyer, H. Huland, G. Sauter, T. Schlomm, Low activated leukocyte cell adhesion molecule expression is associated with advanced tumor stage and early prostate-specific antigen relapse in prostate cancer, Hum. Pathol. 42 (2011) 1946–1952. [10] M. Tachezy, H. Zander, A.H. Marx, F. Gebauer, T. Rawnaq, J.T. Kaifi, G. Sauter, J.R. Izbicki, M. Bockhorn, ALCAM (CD166) expression as novel prognostic biomarker for pancreatic neuroendocrine tumor patients, J. Surg. Res. 170 (2011) 226–232. [11] G. Kristiansen, C. Pilarsky, C. Wissmann, C. Stephan, L. Weissbach, V. Loy, S. Loening, M. Dietel, A. Rosenthal, ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions, Prostate 54 (2003) 34–43. [12] J.A. King, S.F. Ofori-Acquah, T. Stevens, A.B. Al-Mehdi, O. Fodstad, W.G. Jiang, Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator, Breast Cancer Res. 6 (2004) R478–R487.
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