Abstract
Prognosis for parotid carcinomas is not-well defined. The authors have developed models that could be useful to define subgroups of patients with differential risks. Clinical and pathological variables, and immunohistochemically studied MMP-7, MMP-9, MT1-MMP, and VEGF proteins were analysed in 42 patients with parotid gland cancer, regarding disease-specific survival and loco-regional recurrence. A prognostic index (PI) was calculated by combining age, disease stage, squamous cell carcinoma histology, and vascular endothelial growth factor immunoexpression. Based on the values of this PI, patients were classified into three groups: 1.89–4.18 (SPI1); 4.2–7 (SPI2), and >7 (SPI3). Corresponding 5-year survival rates for these groups of patients were of 89%, 47%, and 21%, respectively. Regarding loco-regional recurrence three different patient groups were calculated combining three factors: T, N and grade. Differences amongst them were statistically significant and the estimated hazard ratios were 6.4 and 24.2 for intermediate and poor prognosis, respectively, taking the good prognostic group as reference. The authors identified several significant prognostic factors and they propose two PIs for disease-specific survival and for loco-regional recurrence. They allow for the calculation of death risk and recurrence for a given patient, providing a practical system for clinical use.
Parotid gland carcinomas constitute 2% of all head and neck malignancies , and account for 50–70% of all salivary gland cancers. It is often hard to quantify survival and to determine prognostic factors for these tumours because of their relative rarity, and their diagnosis and management are a surgical challenge. These cancers show considerable diversity in histopathological types, biological behaviour, and tumour grades. Classically, clinical and histological variables have been prognostically relevant, but now the tendency is to think that biological markers may help to improve the prediction of the metastatic potential of salivary gland carcinomas, which will enhance prognosis prediction and provide additional therapeutic information. Several studies have demonstrated over-expression of molecular markers such as EGFR, c-erb-B2, and HER , suggesting the possibility of developing new biologically supported treatments. Metastasis is a multi-step process that appears to be regulated by a variety of gene products including matrix metalloproteinases (MMPs), such as gelatinase B (MMP-9), which plays a role in the degradation of collagen type IV, a key component of basement membrane (BM), and other extracellular matrix (ECM) components . Some authors found no association between MMP-9 and metastasis in salivary gland cancers , whilst others did find an association. MMP-7 (matrilysin) has the capacity to trigger an MMP activation cascade, and can degrade a variety of ECM substrates . Recently, the possible usefulness of this MMP as a prognostic factor in patients with salivary gland carcinomas has been assessed . As for other MMPs thought to play a major role in tumour invasion and metastasis, it has been demonstrated that membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) degrades several components of the ECM and serves as the dominant purveyor of the tissue-invasive activities necessary to support the movement of normal and neoplasic cells through the ECM . Angiogenesis is a biological process crucial for the tumour growth and development of metastasis . In cases of insufficient vasculature, tissues become depleted of oxygen and nutrients, leading to the secretion of angiogenic factors . Vascular endothelial growth factor A (VEGF-A) is the main factor promoting angiogenesis. The authors have shown previously that VEGF expression was significantly correlated with lymph node metastasis, clinical stage, cause-specific survival and local failure-free survival in salivary gland carcinomas.
Prognostic indices for patients with parotid carcinoma based on the combined effects of adverse prognostic factors have been proposed ; but similar patients, affected by tumours with similar clinicopathological variables and undergoing the same treatment, may exhibit widely different prognosis. The convenience of including molecular prognostic markers together with known important clinicopathological variables in the construction of prognostic indices has also been recognized .
The purpose of this study was to identify clinicopathological and molecular markers of disease-specific and recurrence-associated prognostic factors and to develop models that could define subgroups of patients with differential risks.
Materials and methods
This study comprises a retrospective cohort of 42 patients with parotid carcinomas who were diagnosed and treated primarily with surgery at a single centre between January 1987 and December 2000. Inclusion criteria were: surgical treatment with curative intention; complete clinicopathological data; availability of sufficient paraffin-embedded tumour material; histological type with at least five cases; and a follow-up of at least 3 years. Patients with present or previous cutaneous or mucosal head and neck carcinomas were excluded from the study.
Postoperative radiotherapy was applied in the following scenarios : locally-advanced disease; recurrent disease in a non-irradiated site; high-grade histological type; perineural spread; positive or close margins; and neck nodal disease. Chemotherapy was administered in seven cases in a palliative fashion for unresectable relapsing disease, patients not amenable to radiotherapy, and patients with unresectable metastatic disease.
All tumours were staged according to the 2002 AJCC TNM staging system . The grade of the tumour was determined as low or high in the following manner: high grade (high-grade mucoepidermoid carcinoma, adenoid cystic carcinoma, salivary duct carcinoma, adenocarcinoma not otherwise specified, carcinoma in pleomorphic adenoma, small cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma) and low grade (acinic cell carcinoma, polymorphous low-grade adenocarcinoma, epithelial–myoepithelial carcinoma, and basal cell adenocarcinoma) . Mucoepidermoid carcinomas represent a particular case. These tumours are composed of three cell types: mucous producing cells, epidermoid cells, and intermediate cells. Low-grade mucoepidermoid carcinomas produce well-circumscribed unencapsulated masses that contain glandular or microcystic structures lined with well differentiated mucus-producing cells. Solid areas of intermediate and epidermoid cells are infrequent and there is no cellular pleomorphism. High-grade tumours are not well circumscribed and they are characterized by a solid growth pattern, with few glandular or cystic formations. Intermediate and epidermoid cells are more prominent than mucus-producing cells, and cellular pleomorphism and mitosis are easily found. In the present sample, intermediate-grade mucoepidermoid carcinomas were not found.
Clinical outcome was measured by two end-points: death caused by disease or non-treatable disease at the end of the follow-up period; or tumour recurrence. In the authors’ institution, informed consent concerning use of patient information for retrospective studies is not required nor requested.
The antibodies used in this study are listed in Table 1 . Immunohistochemical techniques were performed as previously described . Tissue specimens were fixed in 10% neutral-buffered formalin (pH 7.4) at 4 °C for 72 h, paraffin-embedded, cut into 4-μm sections and transferred onto microscope poly- l -lysine-coated slides. These sections were deparaffined with xylene and dehydrated in graded alcohol. The endogenous peroxidase was blocked by immersion of slides in methanol with 0.3% hydrogen peroxide for 30 min. The sections were rinsed in distilled water and later in phosphate-buffered saline (PBS). Non-specific conjugation was blocked with a solution of 20% rabbit serum (DAKO, Glostrup, Denmark) applied to the sections for 10 min. To establish the optimum concentration for the different antibodies, each antibody was tested using a range of dilutions. The sections were incubated with primary antibodies at different dilutions ( Table 1 ) for 30 min at room temperature and were rinsed twice in PBS for 4 min. Secondary antibody staining was performed using the EnVision polymer technology (K4001, DAKO, Carpinteria, CA) for 30 min according to the manufacturer’s instructions. After washing twice with PBS for 4 min, slides were incubated with 3,3′-diaminobenzidine-tetrahydrochloride in 50 mM Tris–HCl (pH 7.5) containing 0.001% hydrogen peroxide for 5 min, and then lightly counterstained with Mayer’s haematoxylin. One positive and one negative control were included in each batch of immunostained sections. Positive controls were prepared as recommended by the manufacturer ( Table 1 ). For negative controls, the primary antibodies were omitted and replaced with non-immune mouse serum. All slides were independently reviewed by two researchers. The immunostaining was evaluated semi-quantitatively. Staining for MMP-7, MMP-9, MT1-MMP, and VEGF was measured as the percentage of positively stained tumour cells, and scored as negative or positive staining.
| Antibodies | Supplied by | Code | Dilution | Control |
|---|---|---|---|---|
| MMP-7 | Chemicon ® | AB19135 | 1:400 | Skeletal muscle |
| MMP-9 | Neomarkers ® | M887 | 1:700 | Liver cancer |
| MT1-MMP | Chemicon ® | MAB3317 | 1:700 | Smooth muscle |
| VEGF | Neomarkers ® | RB-9031 | 1:100 | Tonsil tissue |
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows (version 11.5, SPSS Inc., Chicago IL, USA). Demographic, clinical and pathological data were summarized using descriptive statistics. Survival analysis was conducted with the Kaplan–Meier method, whilst the log rank test was used to compare survival amongst groups of patients. Survival time was defined as the period from treatment to death, recurrence or last follow-up. Disease-specific survival and loco-regional failure-free survival were calculated from the date of primary treatment for all patients. p values equal or less than 0.05 were considered to be statistically significant. Cox proportional hazard models were also designed to provide hazard ratios (HR) or relative risks for individual variables (univariate analysis) and with all prognostic factors entered in the model together. Afterwards, a backward stepwise likelihood ratio method with threshold values of 0.05 for variable entry and 0.10 for variable exclusion was used. Hazard ratios and associated 95% confidence intervals were computed for statistically significant predictor variables, and used as logarithms of HR. Deaths for causes other than the salivary tumour were considered censored cases.
Results
The main clinical characteristics of the patients included in this study are detailed in Table 2 . Five cases (12%) showed perineural invasion, in 3 of which (7%) vascular invasion occurred. At the conclusion of the study, 22 patients (52%) were alive and free of disease, whilst the remaining 20 patients (48%) had died or showed a non-treatable disease. VEGF immunoreactivity was located in the cytoplasm of the tumour cells in 30 cases (71%). A moderate expression was observed in 16 cases (53%) and intense expression in 14 cases (47%). Protein expression of MMP-7 was located in the cytoplasm of tumour cells, and it was detected in 18 (43%) of 42 cancer tissues. Immunolocalization of matrilysin in carcinoma cells was mainly located in the inner part of tumour islands or cords. Staining in all specimens was restricted to the tumour cells, with no staining of the stroma. Immunostaining for MMP-9 was observed in 27 cases (64%). MMP-9 immunostaining was predominantly localized in tumour cells, and occasionally in the inflammatory stroma cells surrounding tumour islands. The staining pattern irrespective of tumour type was cytoplasmic, diffuse and occasionally granular with tumour cells showing moderate-to-intense positivity, and with no staining in nuclei. Immunostaining for MT1-MMP was seen in 16 of 42 (38%) specimens. Conversely to matrilysin, and similarly to MMP-9, MT1-MMP was immunoexpressed in both tumoral tissue and stroma. Carcinoma cells showed intracytoplasmic staining for MMP-7, MMP-9 and MT1-MMP, but with MT1-MMP, cell membranes were also positively stained.
| Variable | Number (42) | % |
|---|---|---|
| Age (years) mean (SD) (range) (median) | 63 (16) (19–91) (65.5) | |
| Gender | ||
| Male | 21 | 50 |
| Female | 21 | 50 |
| Histology type | ||
| Adenocarcinoma | 10 | 24 |
| Mucoepidermoid carcinoma | 9 | 22 |
| Adenoid cystic carcinoma | 4 | 9 |
| Mixed malignant | 4 | 9 |
| Squamous cell carcinoma | 5 | 12 |
| Acinic cell carcinoma | 10 | 24 |
| Grade | ||
| Low | 16 | 38 |
| High | 26 | 62 |
| T | ||
| T1 + T2 | 32 | 76 |
| T3 + T4 | 9 | 21 |
| Unknown | 1 | 3 |
| N | ||
| N0 | 26 | 62 |
| N+ | 12 | 38 |
| M | ||
| M0 | 33 | 79 |
| M+ | 9 | 21 |
| Stage | ||
| I | 23 | 55 |
| II | 2 | 5 |
| III | 4 | 9 |
| IV | 13 | 31 |
| Status of resection margin | ||
| Free | 36 | 86 |
| Infiltrated | 6 | 14 |
| Recurrence | ||
| No | 24 | 57 |
| Yes | 18 | 43 |
| Radiotherapy | ||
| No | 15 | 36 |
| Yes | 27 | 64 |
| Chemotherapy | ||
| No | 35 | 83 |
| Yes | 7 | 17 |
| Follow-up (months) | ||
| Mean (SD) (range) | 70 (59) (6–260) | |
The mean disease-specific survival time from treatment was 135 months. The 5- and 10-year cause-specific survival rates were 64% and 56%, respectively. As the univariate analysis shows ( Table 3 ), age at diagnosis, gender, N, M, disease stage, histology type, grade, and tumour recurrence were predictive at p < 0.05 for cause-specific survival. In addition, a greater statistically significant number of patients showed positive immunostaining for VEGF (71% vs. 26%) and for MMP-9 (64% vs. 33%). As for the regional control of the disease, the 5- and 10-year regional relapse-free survival rates were 63% and 59%, respectively.
| Prognostic factor | Number | Survival | Survival time (months) Mean, median (95% confidence interval) |
p | |
|---|---|---|---|---|---|
| Number | % | ||||
| Age | |||||
| ≤65 years | 20 | 13 | 65 | 149, 149 (105–192) | 0.01 |
| >65 years | 22 | 9 | 41 | 90, 46 (38–143) | |
| Gender | |||||
| Male | 21 | 8 | 38 | 74, 47 (46–103) | 0.01 |
| Female | 21 | 14 | 67 | 158, 167 (104–211) | |
| T | |||||
| T1 + T2 | 32 | 20 | 62 | 136, 149 (90–181) | 0.06 |
| T3 + T4 | 9 | 2 | 22 | 74, 20 (15–133) | |
| N | |||||
| N0 | 26 | 19 | 73 | 167, 167 (114–219) | 0.0002 |
| N+ | 16 | 3 | 19 | 54, 34 (27–81) | |
| Distant metastasis | |||||
| No | 33 | 22 | 67 | 155, 167 (108–201) | 0.0004 |
| Yes | 9 | 0 | 0 | 46, 46 (18–74) | |
| Stage | |||||
| I + II | 25 | 19 | 76 | 173, 167 (120–226) | <0.00005 |
| III + IV | 17 | 3 | 18 | 52, 34 (26–78) | |
| Histologic type | |||||
| AC | 10 | 5 | 50 | 72, 52 (45–98) | 0.009 |
| MEC | 9 | 3 | 33 | 82, 65 (29–136) | |
| ACC | 4 | 2 | 50 | 158, 149 (83–233) | |
| CCA | 10 | 8 | 80 | 121, 132 (92–150) | |
| MMT | 4 | 3 | 75 | 200 (98–302) | |
| SCC | 5 | 1 | 20 | 23, 8 (3–43) | |
| Grade | |||||
| Low | 16 | 14 | 87 | 185, 132 (100–270) | <0.00005 |
| High | 26 | 8 | 31 | 81, 47 (46–115) | |
| Surgical margin | |||||
| Free | 36 | 21 | 58 | 135, 132 (90–180) | 0.1 |
| Invaded | 6 | 1 | 17 | 68, 46 (15–121) | |
| Recurrence | |||||
| No | 24 | 18 | 75 | 90 (73–106) | 0.04 |
| Yes | 18 | 4 | 22 | 92, 46 (50–134) | |
| VEGF | |||||
| Negative | 11 | 9 | 82 | 193, 167 (125–262) | 0.02 |
| Positive | 30 | 13 | 43 | 97, 81 (60–135) | |
| MMP-7 | |||||
| Negative | 24 | 14 | 58 | 143, 132 (94–192) | 0.1 |
| Positive | 18 | 8 | 44 | 83, 47 (50–115) | |
| MMP-9 | |||||
| Negative | 14 | 12 | 86 | 223 (176–270) | 0.008 |
| Positive | 27 | 10 | 37 | 84, 81 (58–110) | |
| MT1-MMP | |||||
| Negative | 25 | 14 | 56 | 137, 149 (93–181) | 0.2 |
| Positive | 16 | 8 | 50 | 64, 46 (40–88) | |
As shown in the Cox univariate analyses ( Table 4 ) an almost 3% increase in the HR was observed when age was greater than 65 years, compared with younger patients. The squamous cell carcinoma histology showed an almost fourfold increase in HR compared with other histological types. Amongst the other prognostic factors in the univariate analysis, histopathological grade (HR = 7.8 for high degree), regional (HR = 5.5 for neck node positive cases) and distant metastasis (HR = 4.6) showed the highest relative risks. Of all the immunohistochemically studied variables, only immunoreaction for MMP-9 and VEGF was associated with a 5.8 and 4.6 HR, respectively.
| Prognostic factor | Survival | ||
|---|---|---|---|
| HR | 95% CI | p | |
| Age (≤65 years, >65 years) | 2.9 | 1.1–7.5 | 0.02 |
| Gender | 3.2 | 1.2–8.5 | 0.02 |
| Histologic type | |||
| Adenocarcinoma | Reference | 0.03 | |
| Adenoid cystic carcinoma | 0.4 | 0.07–2.3 | 0.3 |
| Malignant mixed tumour | 0.2 | 0.03–2.5 | 0.2 |
| Squamous cell carcinoma | 3.9 | 1.01–15.5 | 0.04 |
| Acinic cell carcinoma | 0.3 | 0.06–1.67 | 0.1 |
| Mucoepidermoid carcinoma | 1.2 | 0.3–4.2 | 0.7 |
| Grade (low, high) | 7.8 | 1.8–33.9 | 0.006 |
| T (T1 + T2, T3 + T4) | 2.4 | 0.9–6.2 | 0.07 |
| N (N0, N+) | 5.5 | 2.05–14.8 | 0.001 |
| Distant metastasis (no, yes) | 4.6 | 1.8–11.7 | 0.001 |
| Stage (I + II, III + IV) | 6.9 | 2.4–19.8 | < 0.0005 |
| Recurrence (no, yes) | 2.6 | 0.9–7.2 | 0.05 |
| Margins (free, positive or close) | 2.2 | 0.7–6.2 | 0.1 |
| VEGF (negative, positive) | 4.6 | 1.05–20.5 | 0.04 |
| MMP-7 (negative, positive) | 2 | 0.8–4.9 | 0.1 |
| MMP-9 (negative, positive) | 5.8 | 1.3–25.7 | 0.01 |
| MT1-MMP (negative, positive) | 1.8 | 0.7–5.07 | 0.2 |
In the Cox proportional hazards model analysis, with the exception of the age of the patients at the time of diagnosis, disease stage, histologic type corresponding to squamous cell carcinoma, and VEGF immunoexpression, the remaining clinical, pathological and immunohistochemical variables were no longer associated with cause-specific survival after adjustment in the Cox multivariate analysis ( Table 5 ). An age over 65 years was associated with a relative risk of 10.7 compared with that of younger patients; the presence of clinical stage III or IV resulted in a 6.6 increase in the HR for cause-specific survival; squamous cell carcinoma histopathology was associated with an HR of 10 compared with the rest of histopathological types, and the HR associated with the final stage increased almost tenfold in those cases in which VEGF immunoexpression was positive.
| Prognostic factor | Cause-specific survival | ||||
|---|---|---|---|---|---|
| B coefficient (ln HR) | SE | HR | HR 95% CI | p | |
| Age | 2.3 | 0.9 | 10.7 | 1.7–67.3 | 0.01 |
| Stage | 1.89 | 0.7 | 6.62 | 1.4–30.6 | 0.01 |
| Histologic type (SCC vs. the rest of types) | 2.3 | 1.02 | 10.13 | 1.3–76.06 | 0.02 |
| VEGF | 2.29 | 1.1 | 9.9 | 1.1–86.6 | 0.03 |
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