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Cell type-dependent biomarker expression in adenoid cystic carcinoma
Biologic and therapeutic implications
Article first published online: 7 SEP 2010
Copyright © 2010 American Cancer Society
Volume 116, Issue 24, pages 5749–5756, 15 December 2010
How to Cite
Bell, D., Roberts, D., Kies, M., Rao, P., Weber, R. S. and El-Naggar, A. K. (2010), Cell type-dependent biomarker expression in adenoid cystic carcinoma. Cancer, 116: 5749–5756. doi: 10.1002/cncr.25541
- Issue published online: 3 DEC 2010
- Article first published online: 7 SEP 2010
- Manuscript Accepted: 28 JUN 2010
- Manuscript Revised: 27 MAY 2010
- Manuscript Received: 9 MAR 2010
- adenoid cystic carcinoma;
- cellular localization;
- epidermal growth factor receptor
Adenoid cystic carcinoma (ACC), a rare and progressive salivary malignancy, is characterized by cellular, morphologic, and clinical heterogeneity. The authors of this report hypothesized that the dual cellular composition of ACC plays an important role in biomarker evaluation, tumor biologic behavior, and response to therapy.
To investigate the differential localization and expression of the c-Kit protein and the epidermal growth factor receptor (EGFR) protein, immunohistochemical analyses were performed on tissue arrays that were constructed from 199 tumors, and the results were correlated with clinicopathologic factors.
c-Kit expression was limited to the inner ductal epithelial cells, whereas EGFR expression was limited mainly to the outer myoepithelial cells in the majority of ACCs with tubular and cribriform patterns. In solid ACCs, c-Kit uniformly was positive, whereas EGFR consistently was negative. A significant statistical correlation was observed between c-Kit expression and a poor 3-year outcome, and EGFR expression was correlated with a better 3-year outcome.
The current findings underscored the importance of cellular subtype localization of biomarkers in the clinical and therapeutic stratification of patients with ACC. Cancer 2010. © 2010 American Cancer Society.
Adenoid cystic carcinoma (ACC) is the second most frequent malignancy of the major and minor salivary glands and comprises approximately 15% to 25% of all carcinomas at these locations.1-3 ACC is formed uniquely of dual epithelial and myoepithelial cells in the conventional tubular and cribriform patterns. Generally, these 2 forms pursue a protracted and progressive course, which can be attributed to the suppressive role of the myoepithelial cells.4-7 Further evidence for this function is provided by the coincident association of the myoepithelial cell loss with the solid form transformation and aggressive behavior.
The role of myoepithelial cells in cellular polarization of the tubular and cribriform patterns and the biologic behavior of ACC has been a focus of our research efforts. We hypothesized that the dual myoepithelial/epithelial structure of the majority of tubular and cribriform ACC and other salivary carcinomas with similar cellular composition underlie, at least in part, their protracted behavior and may have an impact on their therapeutic management. Although several biomarkers have been studied in ACC, including c-Kit (cluster of differentiation 117 [CD117]) and epidermal growth factor receptor (EGFR), to our knowledge, the clinical relevance of the differential cellular localization and expression of these markers in ACC has not been addressed.
Studies of c-Kit (CD117) protein, a member of the class III receptor tyrosine kinase family, have reported overexpression in the majority of ACCs.8-10 Elevated c-Kit expression also has been detected in a variety of other tumors, especially gastrointestinal stromal tumor (GIST), seminoma, and malignant melanoma.11-16 Because c-Kit is a target of the tyrosine kinase inhibitor imatinib mesylate, to which significant treatment response has been achieved in chronic myelogenous leukemia and advanced c-Kit–positive GIST,17-19 interest in its therapeutic potential in patients with ACC has been mounting. Although gain-of-function mutations in exons 9 and 11 of c-Kit and amplification were identified as the critical molecular alterations associated with its overexpression in GIST and seminoma, no such point mutation has been identified in ACC.13, 19, 20
Studies of the EGFR, a 170-kDa transmembrane glycoprotein receptor with tyrosine kinase activity in ACC, also have been conducted. EGFR, a member of importance in the signaling pathways, is implicated in the proliferation, differentiation, and survival of cancer cells. Overexpression of EGFR has been observed in a variety of human cancers, including salivary gland cancers. Numerous anti-EGFR strategies have been applied in retrospective and small prospective clinical trials, including patients with ACC.21-25 To investigate the localization and the differential expression of c-Kit and EGFR proteins and their biologic significance in the assessment of ACC, we performed an immunohistochemistry analysis on 199 ACC samples and correlated the results with clinicopathologic factors.
MATERIALS AND METHODS
Archival, formalin-fixed paraffin blocks of 199 ACCs that were accessioned at The University of Texas M.D. Anderson Cancer Center between 1988 and 2006 were studied. Donor blocks that were selected for the microarray construction were sectioned for hematoxylin-and-eosin preparation and review. After marking spatially different regions for harvesting, two 1.0-mm diameter tissue cores were transferred to the recipient block. Pathologic patterns and the phenotypic expression of c-Kit and EGFR were reviewed and scored independently along with traditional factors, such as sex, age, disease stage, and clinical outcomes.
Immunohistochemistry and Immunoreactivity Analysis
Immunohistochemical analysis for c-Kit and EGFR was performed using the BOND MAX IHC staining protocol from Vision Biosystems (Norwell, Mass) on 4-micrometer paraffin sections of the tissue microarray material. In brief, after dewaxing, washing, and rehydration of the slides through xylene and graded alcohol concentrations, Tris-ethylene diamine tetracetic acid buffer was used for antigen retrieval. Slides were treated subsequently with 3% hydrogen peroxide to block endogenous peroxidase. After incubation with the primary antibodies, c-Kit (Dako; Carpinteria, Calif; 1:100 dilution) and EGFR (Dako; 1:100 dilution), the secondary conjugate antibody was applied; this was followed by chromogen diaminobenzidine and counterstaining with hematoxylin.
Membranous and cytoplasmic staining was scored for c-Kit and EGFR expression in tumor cells. A complete lack of staining was considered negative, and staining was recorded as high (3+), intermediate (2+), or low (1+). The intensity of staining was compared with normal EGFR staining in normal salivary duct control samples; cytoplasmic weak staining (1+) was combined with the negative samples. The expression of c-Kit was recorded as high (3+) if <80% of cells were stained positive, intermediate (2+) if from 30% to 80% of cells were stained positive, and low (1+) if <10% of cells were stained positive.
c-Kit Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) was done on touch preparations of fresh tissue from 27 patients. Hybridization and FISH analyses were done as described previously. To determine the deletion status, 200 individual interphase nuclei were analyzed for each patient.
Correlations within biomarkers and between biomarkers and endpoints were assessed with the Pearson chi-square test or, when there were <10 patients in any cell of a 2 × 2 grid, with the 2-tailed Fisher exact test. Curves describing overall survival were generated by using the Kaplan-Meier product-limit method. The statistical significance of differences between the actuarial curves was tested with the log-rank test. Follow-up was measured from the date of the patient's first appointment at The University of Texas M. D. Anderson Cancer Center for the primary tumor of concern until the date of either last contact or death. Calculated P values <.05 were considered significant. These statistical tests were performed with the assistance of the Statistica (StatSoft, Inc., Tulsa, Okla) and SPSS (SPSS for Windows, SPSS Inc., Chicago Ill) -statistical software applications.
Demographic and Pathologic Findings
The study cohort comprised 106 women and 93 men who ranged in age from 15.9 years to 81.9 years (median age, 51.4 years). Tumor sites included the parotid gland in 29 patients, the hard palate in 28 patients, the maxillary sinus in 26 patients, the submandibular gland in 20 patients, and various minor salivary glands sites in 96 patients. According to American Joint Committee on Cancer staging, 5 patients had stage I disease, 22 patients had stage II disease, 7 patients had stage III disease, and 31 patients had stage IV disease. For 134, patients staging information was not available.
Histopathologic and Clinical Findings
Histopathologically, at least 2 distinctive patterns within and between cores of the same case tumor were observed. A predominant pattern was determined based on the presence of >60% of a given pattern in a tumor. Among the 190 tumors for which a predominant type could be ascertained, 57 tumors were tubular (30%), 109 tumors (57.4%) had predominantly cribriform patterns, and 24 tumors (12.6%) had the solid pattern (mostly devoid of myoepithelial cells). Overall survival plots for the 3 predominant patterns are provided in Figure 1. Five years after presentation, the cumulative survival rate was 73.6% for patients who had tumors with predominant cribriform features, 67.8% for patients who had tubular tumors, and 57.2% for patients who had tumors with a predominantly solid pattern.
Epidermal Growth Factor Receptor and c-Kit Expression
Salivary gland tissues
c-Kit and EGFR were highly expressed in the membranes and/or cytoplasm of salivary ductal cells, whereas moderate cytoplasmic expression was noted in epithelial cells of the intercalated and striated ducts. No immunoreactivity for c-Kit or EGFR was noted in peripheral myoepithelial cells of the intercalated ducts.
Adenoid Cystic Carcinoma tissues
c-Kit expression was limited to the inner ductal cells and was negative in the myoepithelial cells in both tubular and cribriform patterns of ACC (Fig. 2). Restricted c-Kit expression to the inner ductal cells was observed in 24.18% (22 of 91) of tumors with the cribriform pattern, in 22.22% (10 of 45) of tumors with the tubular pattern, and in 71.43% (15 of 21) of the solid type tumors. Paradoxically, EGFR expression was noted mainly in myoepithelial cells and was negative or weakly cytoplasmic positive in ductal cells of both tubular tumors and cribriform tumors. EGFR immunoreactivity was restricted to the myoepithelial cells in 4.44% (2 of 45) of tubular tumors and in 10.99% (10 of 91) of the cribriform tumors. EGFR staining was negative in the solid type tumors (Table 1).
|Marker||Pattern: No. (%)||Total No. (%)|
|EGFR-m||10 (10.9)||2 (4.4)||0 (0)||12 (7.6)|
|c-Kit-d||22 (24.1)||10 (22.2)||15 (71.4)a||47 (29.9)|
|EGFR-m/ c-Kit-d||56 (61.5)||28 (62.2)||1 (4.7)b||85 (54.1)|
|Neither||3 (3.3)||5 (11.1)||5 (23.8)c||13 (0..8)|
Concurrent c-Kit and EGFR immunoreactivity in ductal and myoepithelial cells, respectively, was observed in 62.22% (28 of 45) of tumors with the tubular pattern, in 61.54% (56 of 91) of tumors with the cribriform pattern, and in 4.76% (1 of 21) of solid type tumors. Negative immunoreactivity for both c-Kit and EGFR was observed in 11.11% (5 of 45) of tumors with the tubular pattern, in 3.30% (3 of 91) of tumors with the cribriform pattern, and in 23.81% (5 of 21) of solid type ACCs (Table 1). Not all data points were available for every sample. Only the samples for which the type was known and both markers in both types of cells were scored were included in this tabulation (this accounted for 157 samples). Nine samples showed aberrant marker distribution (Table 2).
|Marker||Pattern: No. (%)||Total No. (%)|
|EGFR-m-d||1 (1.11)||0 (0)||0 (0)||1 (0.64)|
|c-Kit-d-m||1 (1.11)||1 (2.22)||0 (0)||2 (1.28)|
|EGFR-m-d and c-Kit-d||2 (2.22)||2 (4.44)||0 (0)||4 (2.56)|
|EGFR-m-d and c-Kit-d-m||2 (2.22)||0 (0)||0 (0)||2 (1.28)|
|Total||6 (3.82)||3 (1.91)||0 (0)||9 (5.73)|
Fluorescence in situ hybridization for a c-Kit–specific probe performed on touch preparations from 27 tumors (16 cribriform tumors, 6 tubular tumors, and 5 solid tumors) revealed gains in 13 patients (10 cribriform tumors, 2 tubular tumors, and 1 solid tumor), amplification in 1 patient (a cribriform tumor), and normal signals in 13 patients (6 cribriform tumors, 4 solid tumors, and 1 tubular tumor). All specimens had variable immunoreactivity for c-Kit in the epithelial cells (Fig. 3).
The cumulative proportional survival rate for patients was 82.8% at 3 years, 71.7% at 5 years, and 50.9% at 10 years. At last contact, 27 patients (14%) were alive with cancer, 123 patients (62%) patients had no evidence of disease, and 49 patients (25%) were lost to follow-up. Distant metastases were detected in 74 patients (37%). The median follow-up was 62.1 months (range, 1.3-282.7 months).
Concomitant EGFR and c-Kit positivity in ACC patterns with myoepithelial cells was correlated with a better outcome, including a 3-years survival rate of 85.4% and a 5-year survival rate of 75.1% regardless of histologic type (log-rank P = .048). Positive ductal c-Kit expression was associated with 3-year and 5-year survival rates of 68.9% and 58.5%, respectively, regardless of histologic type (log-rank P = .048) (Table 3, Fig. 4).
|EGFR-m and c-Kit-d||79.8||75.9||96.2||69.5||100||100||85.4||75.1|
The current study, the first to our knowledge that addresses the differential cellular localization of biomarkers in ACC, indicates that c-Kit expression is limited only to inner epithelial cells. However, expression of EGFR was detected mainly in myoepithelial cells in both the tubular and cribriform phenotypes, but it also was observed in inner ductal cells in some tumors. In the solid form in which myoepithelial cells are is lost, uniform positive staining for c-Kit and lack of EGFR staining were observed. These findings lend credence to the hypothesis that the histogenetic differences between myoepithelial cells and epithelial cells may underlie the biologic heterogeneity and indirectly influence the response to therapy in patients with these tumors.
The incidence of c-Kit expression in our study is in agreement with the majority of previous studies of ACC.8-10, 26 Only 1 of those studies reported the localization of c-Kit in ACC ductal cells10 and its diffuse expression in tumors with the solid pattern. Although the underlying mechanism for the epithelial cell expression of c-Kit in ACC remains to be defined, we contend that the restricted expression to the epithelial cells should be considered in the evaluation of this marker. In the current study, c-Kit expression in the epithelial cells of ACC was associated with a poor 3-year outcome and was independent of the histologic patterns of these tumors.
Studies of c-Kit in GIST and seminoma—tumors with uniform cellular composition—have demonstrated that gain-of-function mutations of the c-kit locus result in overexpression of the protein.13, 27 c-Kit overexpression in the absence of activating point mutations also has been described in various other tumor types, including neuroblastoma, uterine sarcoma, and thymic carcinoma.28, 29 These findings indicate that other genetic, epigenetic, and biologic events and gene copy numbers (eg, in glioblastoma and small cell lung cancers) may be involved in c-Kit regulation. We also reported the amplification of 4q11-12 in 1 patient,20 and a small number of tumors have increased copy numbers of this gene.
Although our analysis demonstrated strong EGFR expression in the myoepithelial cells of tumors with tubular and cribriform patterns, the ductal cells also expressed this marker in some tumors. The reported incidence of EGFR expression in studies of ACC ranges from negative to 70.30-32 In those studies, however, the localization of EGFR expression and other markers was not reported. Only a study of lung ACC reported the dominance of localization to the myoepithelial component.33 Correlations between EGFR or c-Kit expression and tumor size, lymph node status, or tumor stage are rare and inconclusive. In our study, EGFR expression was correlated with better 3-year survival.
The differential expression of c-Kit and EGFR in ACC in our study underscores the biologic heterogeneity of ductal and myoepithelial cells and the impact of such finding on the stratification of patients for a targeted response to imatinib therapy. We contend that this may have played a role in the inconclusive effect of imatinib on the clinical course of ACC. A recent study reported that the receipt of neoadjuvant imatinib by 2 patients who had initially unresectable ACC resulted in significant tumor regression.34 In contrast, the results from a phase 2 study of imatinib therapy for the treatment of advanced ACC produced no objective responses in all 16 patients after 2 cycles.35, 36 However, we reason that the lack of response may have been caused by cellular heterogeneity, because the response can be limited to c-Kit expression in the epithelial cell component but not in the myoepithelial cell component.
In addition to c-Kit, the finding of EGFR expression in ACC indicates that this marker can be an attractive target for therapy.32 It has been demonstrated that EGFR contributes to tumorigenesis in several human carcinomas by blocking apoptosis and promoting angiogenesis. Anti-EGFR–targeted therapies, including cetuximab and erlotinib, have been used in clinical trials in solid tumors.22, 37 The finding that EGFR was expressed mainly in myoepithelial cells and was correlated with better survival in patients ACC may be associated in part with the tumor-suppressive effects and protracted clinical course of tumors that have myoepithelial involvement. Moreover, such a pattern of EGFR expression may have an impact on the selection of patients for anti-EGFR therapy. In that context, patients with solid type tumors may be excluded from such treatment along with those who have tumors with tubular and cribriform patterns composed only of epithelial cells.
In conclusion, the current study has provided evidence for a new therapeutic strategy in ACC based on the differential expression of markers in ductal cells and myoepithelial cells in which combined therapy may benefit patients who have tumors with tubular and cribriform patterns, and patients with solid type tumors may be responsive to a single agent (eg, an anti-c-Kit agent). Therefore, we attribute the lack of progress in clinical trials of ACC, in addition to including other phenotypic carcinomas, to the differential response to therapy in myoepithelial cells and epithelial tumor cells. This is supported by evidence for a less aggressive and modulating role of myoepithelial cells in mammary and salivary carcinomas and the generally low malignant nature of salivary and breast carcinomas with myoepithelial participation. It may be better to base future therapeutic trials of these patients on biomarker stratification and the cellular composition of their tumors.
CONFLICT OF INTEREST DISCLOSURES
The study is supported in part by Award Number U01DE019756 from the NIH National Institute of Dental and Craniofacial Research (NIDCR) and the NIH Office of Rare Diseases Research (ORDR), the Head and Neck SPORE program and the Kenneth D. Muller professorship, and the NCI CA16672 grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.