Seongyeol Park and Soo Jeong Nam contributed equally to this work.
The purpose of this study was to evaluate potential prognostic factors in patients with adenoid cystic carcinoma (ACC).
A total of 68 patients who underwent curative surgery and had available tissue were enrolled in this study. Their medical records and pathologic slides were reviewed and immunohistochemistry for basic fibroblast growth factor, fibroblast growth factor receptor (FGFR) 2, FGFR3, c-kit, Myb proto-oncogene protein, platelet-derived growth factor receptor beta, vascular endothelial growth factor (VEGF), and Ki-67 was performed. Univariate and multivariate analysis was performed for determination of disease-free survival (DFS) and overall survival (OS).
In univariate analyses, primary site of nasal cavity and paranasal sinus (p=0.022) and Ki-67 expression of more than 7% (p=0.001) were statistically significant factors for poor DFS. Regarding OS, perineural invasion (p=0.032), high expression of VEGF (p=0.033), and high expression of Ki-67 (p=0.007) were poor prognostic factors. In multivariate analyses, primary site of nasal cavity and paranasal sinus (p=0.028) and high expression of Ki-67 (p=0.004) were independent risk factors for poor DFS, and high expression of VEGF (p=0.011) and Ki-67 (p=0.011) showed independent association with poor OS.
High expression of VEGF and Ki-67 were independent poor prognostic factors for OS in ACC.
Adenoid cystic carcinoma (ACC) is uncommon malignancies, which commonly arise in the salivary glands [
ACC is characterized by unpredictable growth and extensive perineural invasion. Typically the natural course of ACC is slow; however, local recurrence and hematogenous spread to the lungs often occur during the course of the disease [
Due to its rarity, the molecular biology of ACC has not been well-described [
A previous study reported that fibroblast growth factor (FGF), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor beta (PDGFR-beta) represented DNA copy number gain in ACC by microarray-based comparative genomic hybridization [
The aim of this study was to evaluate the prognostic value of potential biomarkers related to ACC.
The medical records of 188 patients diagnosed with ACC at Seoul National University Hospital between 1990 and 2012 were reviewed. Among these patients, 68 patients who underwent curative surgery and had available tissue were enrolled in this study.
This study was approved by the Institutional Review Board of Seoul National University Hospital (IRB approval No. H-1109-114-379). Demographics, clinical and pathologic data, and treatment-related factors with regard to recurrence and patient survival were collected from medical records. Sixteen patients (24%) were followed up until death, and the median follow-up period was 68 months (range, 4 to 263 months).
Core tissues (2 mm in diameter) were taken from representative formalin-fixed paraffin-embedded tissue blocks and tissue microarrays were constructed for further immunohistochemical analysis. The antibodies used were basic FGF (bFGF; 1:100, Calbiochem, San Diego, CA), FGFR2 (H2263-M01, 1:3,000, Abnova, Taipei, Taiwan), FGFR3 (SC-13121, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA), c-kit (A2502, 1:200, DAKO, Carpinteria, CA), MYB (#1792-1, 1:80, Epitomics, Burlingame, CA), PDGFR-beta (SC-713, 1:100, Santa Cruz Biotechnology), VEGF (SC-7269, 1:1,000, Santa Cruz Biotechnology), and Ki-67 (M7240, 1:100, DAKO).
Immunostaining was evaluated semi-quantitatively for intensity (0, negative; +1, weak positive; +2, moderate positive; and +3, strong positive) and extent (0, 0%; +1, 1%-25%; +2, 26%-50%; +3, 51%-75%; and +4, 76%-100%) by two experienced pathologists (S.J.N., Y.K.J.); normal salivary gland samples were used as controls. Score was determined by multiplying extent and intensity. Positivity of expression was defined as a score of 4 or more, and high expression was defined as a score of 8 or more. Conversely, c-kit was graded only by extent (0 to +4) because its expression was strong enough in all positive specimens, and positivity was defined as a grade of 2 or more [
The immunostained slides for Ki-67 were submitted to virtual microscope scanning under high-power magnification (×200) using ScanScope CS2 eSlide (Aperio Technologies, Vista, CA). Estimation of Ki-67 expression was based on the proportion of positive cells in all tumor cells using the nuclear v9 algorithm of ImageScope software (Aperio Technologies). Ki-67 was considered positive when the tumor cells showed strong nuclear staining intensity.
Statistical analyses of categorical variables were performed using Pearson’s χ2 test or Fisher exact probability test where appropriate. The median duration of survival was calculated using the Kaplan-Meier method, and comparisons between groups were made using the log-rank tests. To determine the important prognostic factors, Cox proportional hazards regression models were used in univariate and multivariate analyses. A two-sided value of p < 0.05 was considered statistically significant. All analyses were performed using SPSS for Windows ver. 20.0 (IBM Co., Armonk, NY).
Of the 68 patients, 26 patients were male and 42 were female, with median age of 55 years (range, 26 to 84 years). Primary sites were major salivary glands in 36 cases (53%), nasal cavity and paranasal sinuses in 16 (24%), oral cavity and tongue in seven (10%), lung and trachea in four (6%), and other sites in five (7%). Other sites included the lacrimal gland, Bartholin’s gland, and external auditory canal. Twenty-three patients (34%) had perineural invasion and 39 (57%) had involvement of resection margins. Local recurrence was detected in 21 patients (31%), regional recurrence in three (4%), distant metastases in 35 (51%), and any kind of recurrence in 39 (57%). Of patients with distant metastases, 32 patients (47%) had lung metastasis, nine (13%) had liver metastasis, six (9%) had bone metastasis, and two (3%) had central nervous system metastasis.
Analysis of patients was based on clinicopathological factors, including sex, age, primary site, local treatment modality including postoperative radiotherapy, systemic chemotherapy, perineural invasion, and positive resection margins. Survival analysis was performed in each subgroup using the Cox proportional hazards regression model. Among these clinicopathological variables, primary site of nasal cavity and paranasal sinus was a poor prognostic factor for disease-free survival (DFS; hazard ratio [HR], 2.34; 95% confidence interval [CI], 1.13 to 4.84; p=0.022). Perineural invasion was the only significant prognostic factor of poor overall survival (OS; HR, 3.05; 95% CI, 1.10 to 8.44; p=0.032) (
In immunohistochemistry, overexpression of bFGF was observed in 48 patients (71%), c-kit in 52 (77%), FGFR2 in six (9%), FGFR3 in four (6%), MYB in 24 (35%), PDGFR-beta in 43 (63%), and VEGF in 62 (91%). The median Ki-67 value was 4% (range, 0% to 33%) (
In univariate analysis, positive expression with a score of 4 or more for bFGF, FGFR2, MYB, PDGFR-beta, and VEGF and a score of 2 or more for c-kit did not show correlation with DFS and OS (
In multivariate analysis, primary site of nasal cavity and paranasal sinus (HR, 2.21; 95% CI, 1.09 to 4.50; p=0.028) and Ki-67 expression (HR, 3.05; 95% CI, 1.43 to 6.54; p=0.004) were independent risk factors for poor DFS. High expression of VEGF (HR, 5.44; 95% CI, 1.48 to 19.98; p=0.011) and Ki-67 (HR, 4.83; 95% CI, 1.44 to 16.21; p=0.011) were independently significant factors of poor OS. However, perineural invasion was not statistically significant for OS (HR, 2.90; 95% CI, 1.00 to 8.41; p=0.051) (
The goal of the current study was to identify prognostic markers of ACC by evaluating the relationship between clinicopathological and immunohistochemical data and DFS and OS. The markers bFGF, c-kit, FGFR2, FGFR3, MYB, PDGFR-beta, VEGF, and Ki-67 were evaluated by immunohistochemistry. In univariate analysis, nasal cavity and paranasal sinus as primary site and Ki-67 expression of more than 7% were risk factors for poor DFS, and presence of perineural invasion and high expression of VEGF and Ki-67 were significant prognostic factors for poor OS. Among them, high expression of VEGF and Ki-67 were independently significant prognostic factors for OS in multivariate analyses.
ACC has diverse primary sites and there are varying levels of difficulty of operation according to primary sites. Ciccolallo et al. [
In the current study, perineural invasion was observed in 23 patients (34%), and it was the only significant prognostic factor for OS among the clinicopathological values evaluated. Its importance as a poor prognostic factor is well-established in the literature [
VEGF, a critical factor in tumor angiogenesis, has been widely assessed in various types of cancer. Association of VEGF expression with poor prognosis of ACC has been reported in a few studies [
Ki-67 is associated with cellular proliferation in tumor progression. Several studies identified high expression of Ki-67 as a negative prognostic factor in salivary gland carcinomas [
One of the most studied biomarkers of ACC is the translocation between
PDGFR-beta expression is known to be a poor prognostic factor in breast cancer [
Most studies conducted in the past analyzed the utility of immunohistochemical markers with respect to ACC invasiveness and recurrence. However, the current study was conducted in a large population and investigated the correlation between several immunohistochemical markers and survival, which is more critical for ACC prognosis. We acknowledge that there are some limitations to this study, including the lack of validating the immunohistochemical cut-off values used, and the fact that this is a retrospective study.
In conclusion, high expression of VEGF and high expression of Ki-67 are independent prognostic factors of poor OS in ACC. Therefore, more aggressive and differentiated treatment should be provided to patients who show high expression of these markers. Conduct of further prospective studies in larger populations will be necessary in order to confirm these prognostic factors and to elucidate appropriate treatment modalities.
Conflict of interest relevant to this article was not reported.
This study was supported by SNUH Research Fund (grant no. 04-2013-0760 and 30-2013-0070). We especially thank Ju Yon Kim for her data management.
Immunohistochemical staining of adenoid cystic carcinoma. (A) Low expression of vascular endothelial growth factor (VEGF). (B) High expression of VEGF. (C) Low expression of Ki-67. (D) High expression of Ki-67 (A-D, ×200).
Kaplan-Meier curve for disease-free survival by vascular endothelial growth factor expression (A) and Ki-67 expression (B). Comparisons were made using the log-rank test.
Kaplan-Meier curve for overall survival by vascular endothelial growth factor (VEGF) expression (A), Ki-67 expression (B), and VEGF and/or Ki-67 expression (C). Comparisons were made using the log-rank test.
Clinicopathological features and univariate Cox regression analysis of 68 patients
Characteristic | No (%) | DFS |
OS |
||
---|---|---|---|---|---|
HR | p-value | HR | p-value | ||
Male | 26 (38.2) | 1 | 1 | 0.881 | |
Female | 42 (61.8) | 0.78 | 0.463 | 1.09 | |
≤ 45 | 17 (25.0) | 1 | 1 | 0.658 | |
> 45 | 51 (75.0) | 1.55 | 0.251 | 1.30 | |
Salivary gland | 36 (52.9) | 1 | 1 | ||
Nasal cavity, paranasal sinus | 16 (23.5) | 2.34 | 0.022 | 1.61 | 0.420 |
Tongue, oral cavity | 7 (10.3) | 2.01 | 0.146 | 0.81 | 0.841 |
Lung, trachea | 4 (5.9) | 0.33 | 0.280 | 1.44 | 0.738 |
Others |
5 (7.4) | 1.00 | 0.995 | 1.01 | 0.994 |
Operation with PORT | 49 (72.1) | 0.96 | 0.919 | 2.16 | 0.311 |
Operation without PORT | 19 (27.9) | 1 | 1 | ||
Yes | 11 (16.2) | - |
- |
2.32 | 0.116 |
No | 57 (83.8) | - | 1 | ||
Yes | 23 (33.8) | 1.26 | 0.501 | 3.05 | 0.032 |
No | 45 (66.2) | 1 | 1 | ||
Positive | 39 (57.4) | 0.97 | 0.913 | 1.49 | 0.456 |
Negative | 29 (42.6) | 1 | 1 |
DFS, disease-free survival; OS, overall survival; HR, hazard ratio; PORT, postoperative radiotherapy.
Bartholin’s gland, external auditory canal, and lacrimal gland,
There was no perioperative chemotherapy.
Positive expression of molecular markers and univariate Cox regression analysis of disease-free survival and overall survival in adenoid cystic carcinoma
Variable | Score | No. (%) | DFS |
OS |
||||
---|---|---|---|---|---|---|---|---|
HR | 95% CI | p-value | HR | 95% CI | p-value | |||
bFGF | < 4 | 20 (29.4) | 1 | 1 | ||||
≥ 4 | 48 (70.6) | 0.76 | 0.39 to 1.49 | 0.424 | 1.25 | 0.35 to 4.47 | 0.737 | |
c-kit | < 2 | 16 (23.5) | 1 | 1 | ||||
≥ 2 | 52 (76.5) | 0.94 | 0.46 to 1.94 | 0.872 | 0.75 | 0.23 to 2.43 | 0.632 | |
FGFR2 | < 4 | 62 (91.2) | 1 | 1 | ||||
≥ 4 | 6 (8.8) | 1.45 | 0.51 to 4.10 | 0.488 | 1.86 | 0.40 to 8.68 | 0.432 | |
FGFR3 | < 4 | 64 (94.1) | 1 | 1 | ||||
≥ 4 | 4 (5.9) | 3.26 | 1.13 to 9.44 | 0.029 | 0.94 | 0.12 to 7.22 | 0.952 | |
MYB | < 4 | 44 (64.7) | 1 | 1 | ||||
≥ 4 | 24 (35.3) | 1.02 | 0.51 to 2.02 | 0.959 | 1.14 | 0.36 to 3.62 | 0.831 | |
PDGFR-beta | < 4 | 25 (36.8) | 1 | 1 | ||||
≥ 4 | 43 (63.2) | 1.10 | 0.57 to 2.13 | 0.770 | 2.43 | 0.68 to 8.62 | 0.171 | |
VEGF | < 4 | 6 (8.8) | 1 | 1 | ||||
≥ 4 | 62 (91.2) | 1.96 | 0.60 to 6.40 | 0.264 | 2.53 | 0.33 to 19.39 | 0.371 |
DFS, disease-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; bFGF, basic fibroblast growth factor; FGFR2, fibroblast growth factor receptor 2; FGFR3, fibroblast growth factor receptor 3; MYB, Myb proto-oncogene protein; PDGFR-beta, platelet-derived growth factor receptor beta; VEGF, vascular endothelial growth factor.
High expression of molecular markers and univariate Cox regression analysis of disease-free survival and overall survival in adenoid cystic carcinoma
Variable | Score | No. (%) | DFS |
OS |
||||
---|---|---|---|---|---|---|---|---|
HR | 95% CI | p-value | HR | 95% CI | p-value | |||
bFGF | < 8 | 42 (61.8) | 1 | 1 | ||||
≥ 8 | 26 (38.2) | 0.85 | 0.44 to 1.62 | 0.618 | 1.25 | 0.44 to 3.51 | 0.673 | |
c-kit | < 4 | 51 (75.0) | 1 | 1 | ||||
≥ 4 | 17 (25.0) | 1.12 | 0.52 to 2.45 | 0.771 | 1.13 | 0.25 to 5.13 | 0.876 | |
FGFR2 | < 8 | 67 (98.5) | 1 | 1 | ||||
≥ 8 | 1 (1.5) | 15.98 | 1.79 to 143.04 | 0.013 | 62.50 | 3.91 to > 99.99 | 0.003 | |
FGFR3 | < 8 | 67 (98.5) | 1 | 1 | ||||
≥ 8 | 1 (1.5) | 4.09 | 0.54 to 31.10 | 0.174 | 0.05 | < 0.01 to > 99.99 | 0.883 | |
MYB | < 8 | 60 (88.2) | 1 | 1 | ||||
≥ 8 | 8 (11.8) | 1.13 | 0.40 to 3.19 | 0.820 | 0.04 | < 0.01 to 44.54 | 0.370 | |
PDGFR-beta | < 8 | 55 (80.9) | 1 | 1 | ||||
≥ 8 | 13 (19.1) | 1.14 | 0.52 to 2.49 | 0.736 | 2.08 | 0.70 to 6.20 | 0.189 | |
VEGF | < 8 | 29 (42.6) | 1 | 1 | ||||
≥ 8 | 39 (57.4) | 1.31 | 0.69 to 2.48 | 0.403 | 3.45 | 1.11 to 10.71 | 0.033 | |
Ki-67 | < 7% | 50 (73.5) | 1 | 1 | ||||
≥ 7% | 18 (26.5) | 3.25 | 1.58 to 6.68 | 0.001 | 4.47 | 1.52 to 13.18 | 0.007 |
DFS, disease-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; bFGF, basic fibroblast growth factor; FGFR2, fibroblast growth factor receptor 2; FGFR3, fibroblast growth factor receptor 3; MYB, Myb proto-oncogene protein; PDGFR-beta, platelet-derived growth factor receptor beta; VEGF, vascular endothelial growth factor.
Multivariate Cox regression analysis of diseasefree survival and overall survival in adenoid cystic carcinoma
Variable | Category | HR | 95% CI | p-value |
---|---|---|---|---|
Nasal area |
No | 1 | ||
Yes | 2.21 | 1.09 to 4.50 | 0.028 | |
Perineural invasion | No | 1 | ||
Yes | 1.29 | 0.65 to 2.55 | 0.460 | |
Resection margin | No | 1 | ||
Yes | 0.84 | 0.39 to 1.78 | 0.641 | |
PORT | No | 1 | ||
Yes | 1.09 | 0.47 to 2.52 | 0.837 | |
VEGF | < 8 | 1 | ||
≥ 8 | 1.31 | 0.67 to 2.56 | 0.435 | |
Ki-67 | < 7% | 1 | ||
≥ 7% | 3.05 | 1.43 to 6.54 | 0.004 | |
Perineural invasion | No | 1 | ||
Yes | 2.90 | 1.00 to 8.41 | 0.051 | |
VEGF | < 8 | 1 | ||
≥ 8 | 5.44 | 1.48 to 19.98 | 0.011 | |
Ki-67 | < 7% | 1 | ||
≥ 7% | 4.83 | 1.44 to 16.21 | 0.011 |
HR, hazard ratio; CI, confidence interval; DFS, disease-free survival; PORT, postoperative radiotherapy; VEGF, vascular endothelial growth factor; OS, overall survival.
Primary sites including nasal cavity and paranasal sinus.