Background: Management of N0 neck in patients with head and neck squamous cell carcinoma (HNSCC) remains a subject of continued debate. Prognostic biomarkers might provide useful information for treatment selection and adjustment. Objective: To evaluate the prognostic relevance of VEGF-A and Ki-67 expression to types of neck management. Methods: This prospective study included 140 patients with HNSCC. Tumor expression of VEGF-A and Ki-67 was measured by immunohistochemistry. Based on tumor size and site criteria, 88 patients with N0 neck were categorized as high, intermediate and low risk of subclinical neck diseases and accordingly treated by elective neck dissection (END), irradiation (ENI) and observation. Adjuvant treatment was given to tumor with close or positive margins. A multivariate Cox regression model was used to identify prognostic factors. Impact of biomarker expression, treatment type and risk category on disease-specific survival (DSS) in the setting of N0 neck were evaluated by Kaplan-Meier survival and adjusted hazard ratio (HR). Results: Coexpression of VEGF-A and Ki-67 (HR = 2.351, p = 0.021) and positive node (HR = 2.301, p = 0.009) were independent prognostic factors for HNSCC. In the setting of N0 neck, marker coexpression has an HR of 4.97 ( p = 0.004) independent of treatment modalities ( p = 0.069) and risk categories ( p = 0.971). Alternatively, neither marker expression was predictive of a better treatment outcome for END compared to ENI, as suggested by the odds of patients being survived 15.4 times greater ( p = 0.01) and the 5-year DSS rates of 85.1% versus 44.7% ( p = 0.008). Conclusion: Coexpression of VEGF-A and Ki-67 is a suggestion of tumor microinvasiveness in addition to risk of lymph node metastasis and may indicate the need of adjuvant treatment despite negative tumor margins. Neither marker expression serves an indicator for the selection of END over ENI in neck management.
Head and neck squamous cell carcinoma (HNSCC) has a high propensity to invade lymph nodes in the cervical region before the development of distant metastasis [
In management of patients with cN0 neck, there are three policies advocated: a watchful waiting policy for those with a risk < 20%; elective neck dissection (END) or elective neck irradiation (ENI) for a risk ≥ 20%; and neck treatment plus adjuvant therapy for the high risk patients [
Pretreatment risk stratification of patients may provide information that helps selecting the appropriate modality in neck management for a particular risk category [
Recent advances in molecular biology have revealed that primary tumor is predisposed to metastasize [
This investigation is the continuum of a prospective study on the use of VEGF-A and Ki-67 in prediction of LN metastasis in HNSCC [
This prospective study cohort has been previously described [
To evaluate whether VEGF-A and Ki-67 expression could serve as a significant prognostic indicator for the present series of patients with surgery treated tumor of the head and neck, demographic (age and gender), and clinicopathological factors (tumor grade and location, T stage and nodal status) were included in survival analysis. Of a total of 147 patients included previously, only 140 were eligible for study because of the availability of complete data for survival analysis. Of these 140 patients, 52 (37.1%) had pathologically confirmed positive neck nodes and 88 (62.9%) had negative nodes either clinically defined or pathologically confirmed. Demographic, clinicopathological characteristics and biomarker expression in patients with positive and negative necks are presented in
Variable | Total, n | Node negative n (%) | Node positive n (%) | p |
---|---|---|---|---|
Age at diagnosis ≤64 years >64 years | 73 67 | 43 (58.9) 45 (67.2) | 30 (41.1) 22 (32.8) | 0.40 |
Sex Male Female | 112 28 | 73 (34.8) 15 (46.4) | 39 (65.2) 13 (53.6) | 0.36 |
Tumor site Oral cavity Pharynx Larynx | 57 27 56 | 40 (70.2) 3 (11.1) 45 (80.4) | 17 (29.8) 24 (88.9) 11 (19.6) | <<0.0001 |
Differentiation Well Moderate and poor | 89 51 | 64 (71.9) 24 (47.1) | 25 (28.1) 27 (52.9) | 0.014 |
T stage T1, 2 T3, 4 | 62 78 | 46 (74.2) 42 (53.8) | 16 (25.8) 36 (46.2) | 0.021 |
VEGF-A High > 2.74 Low ≤ 2.74 | 55 85 | 25 (45.5) 63 (74.1) | 30 (54.5) 22 (25.9) | 0.001 |
Ki-67 High > 58.61% Low ≤ 58.61% | 55 85 | 31 (56.4) 58 (68.2) | 24 (43.6) 27 (31.8) | 0.21 |
Combined expression Both high Either high Neither high | 27 59 54 | 11 (40.7) 36 (61) 41 (75.9) | 16 (59.3) 23 (39) 13 (24.1) | 0.008 |
Eighty-eight patients with N0 neck were grouped as high risk, intermediate risk and low risk of occult neck diseases according to risk definition defined by Mendenhall et al. [
Elective treatment of the neck was performed in patient with a risk of subclinical neck disease ≥ 20% [
Risk category | Occult neck diseases | T | Primary tumor site | n (%) |
---|---|---|---|---|
High | >30% | T3-4 | Pyriform sinus and base of tongue | 3 (8.3%) |
n = 36 | T3-4 | Lip, buccal mucosa, tongue and hard palate | 11 (30.6%) | |
T2-4 | Supraglottis | 10 (27.8%) | ||
T4 | Glottis | 11 (30.5%) | ||
T3 | Subglottis | 1 (2.8%) | ||
Intermediate | 20% - 30% | T2 | Lip, buccal mucosa and hard palate | 3 (10.3%) |
n = 29 | T1-2 | Tongue | 18 (62.1%) | |
T3 | Glottis | 8 (27.6%) | ||
Low | <20% | T1 | Lip and floor of mouth | 8 (34.78%) |
n = 23 | T1-2 | Glottis | 14 (60.87%) | |
T2 | Subglottis | 1 (4.35%) |
High risk | Intermediate risk | Low risk | p | |
---|---|---|---|---|
n = 36 | n = 29 | n = 23 | ||
VEGF-A and Ki-67 | 0.002 | |||
Both high | 2 (5.6) | 5 (17.2) | 4 (17.4) | |
Either high | 23 (63.9) | 5 (17.2) | 8 (34.8) | |
Neither high | 11 (30.6) | 19 (65.5) | 11 (47.8) | |
Neck management | ||||
END + Adj Tx | 17 (47.2) | ? | ? | 0.000 |
ENI | 11 (30.6) | 15 (51.7) | 7 (30.4) | |
END | 8 (22.2) | 14 (48.3) | ? | |
Observation | ? | ? | 16 (69.6) |
END + Adj Tx: Elective neck dissection + adjuvant treatment; ENI: Elective neck irradiation; END: Elective neck dissection.
The follow-up started after treatment completion in every 2 months during the first year, every 3 months in the second and every 6 months thereafter. Disease-specific survival (DSS) was the endpoint of outcome for this study. Time-to-event was defined as the duration since the date of diagnosis to the date of tumor-related death or the date of last follow-up for patients who were still alive. Data were censored for patients who were alive at the end of study; lost to follow-up; dead from causes not attributable to cancer. This study had a median follow-up time of 44 months (range, 1 - 102.1 months).
The expression of Ki-67 was determined by the percentage of positive tumor cells in hot spot area. For VEGF-A, a score varying from 0 to 4 was graded on the basis of stained intensity and percentage of cell stained [
Comparison of categorical data between groups was conducted by χ2 or Fisher Exact test as appropriate. Survival analyses were performed using Predictive Analytics Software (PASW) statistics 18 (SPCC Inc., Chicago, IL, USA). To identify independent prognostic factors for this patient cohort, univariate and multivariate analyses were carried out using Cox proportional hazard regression model. Besides biomarker expression, the survival analysis also included factors generally known to influence survival of head and neck cancer patients. These factors are age at diagnosis, sex, tumor site, tumor differentiation, T stage and nodal status [
In the setting of N0 neck, impact of risk category, type of neck management and biomarker expression on DSS was assessed by Kaplan-Meier survival curve, and multivariate Cox regression model. All statistical tests were two-tailed and p value < 0.05 was considered significant.
The median follow-up time for this study was 44 months (range, 1 - 102.1 months). During follow-up periods, a total of 70 patients died: 60 due to HNSCC, 10 from tumor-unrelated causes such as diseases of the lung and liver, heart attack, old age, etc; 16 patients were lost to follow-up; 44 patients were alive at the time of last contact. The impact on disease-specific survival (DSS) was evaluated for a number of potential influencing factors including age at diagnosis, sex, primary tumor site, tumor differentiation, T stage, nodal status, and biomarker expression using univariate and multivariate Cox proportion hazard regression models. Results of the analyses are presented in
In univariate survival analysis, combined marker expression (p = 0.001) and modality of neck management (p = 0.014) were significantly affecting DSS in contrasting to the nonsignificant effect of the risk category (p = 0.618) (Figures 1-3). Multivariate Cox regression analysis revealed an overall significant p level of 0.013 for VEGF-A
Variable | Univariate | Multivariate Multivariate | ||
---|---|---|---|---|
HR (95% CI) | p | HR (95% CI) | p | |
Age at diagnosis >64 years ≤64 years | 1.086 (0.654 - 1.801) 1 | 0.751 | 1.289 (0.765 - 2.173) 1 | 0.340 |
Sex Male Female | 0.848 (0.458 - 1.568) 1 | 0.599 | 1.253 (0.653 - 2.402) 1 | 0.498 |
Tumor site Oral and pharynx Larynx | 1.836 (1.063 - 3.169) 1 | 0.029 | 1.650 (0.880 - 3.092) 1 | 0.118 |
Differentiation Moderate and poor Well | 1.11 (0.66 - 1.869) 1 | 0.694 | 0.705 (0.401 - 1.238) 1 | 0.223 |
T stage T3,4 T1,2 | 1.43 (0.85 - 2.408) 1 | 0.178 | 1.239 (0.701 - 2.189) 1 | 0.461 |
Nodal status N+ N0 | 3.022 (1.809 - 5.049) 1 | 0.000 | 2.301 (1.231 - 4.304) 1 | 0.009 |
VEGF-A High > 2.74 Low ≤ 2.74 | 1.932 (1.161 - 3.215) 1 | 0.011 | 1.435 (0.807 - 2.553) 1 | 0.219 |
Ki-67 High > 58.61 Low ≤ 58.61% | 1.744 (1.05 - 2.898) 1 | 0.032 | 1.559 (0.929 - 2.615) 1 | 0.093 |
Combined expression Both high Either high Neither high | 2.987 (1.569 - 5.687) 1.241 (0.665 - 2.314) 1 | 0.001 0.498 | 2.357 (1.14 - 4.873) 1.187 (0.559 - 2.353) 1 | 0.021 0.624 |
HR: Hazard ratio; CI: Confidence interval.
and Ki-67 expression. In relative to tumor with neither marker expression, only coexpression of VEGF-A and Ki-67 was observed with a significant HR of 4.97 (p = 0.004). For the modality of neck management, the impact was at a marginally significant level with a p value of 0.069. In comparing different types of neck treatment to observation, significant HR of 8.737 (p = 0.036) was obtained for neck treatment by ENI, and a marginally significant HR of 6.043 (p = 0.098) for END + adjuvant treatment (
Variable | HR | 95% CI | p | |
---|---|---|---|---|
Lower | Upper | |||
VEGF-A and Ki-67 | 0.013 | |||
Both high | 4.97 | 1.669 | 14.804 | 0.004 |
Either high | 1.659 | 0.651 | 4.41 | 0.28 |
Neither high | 1 | ? | ? | ? |
Neck management | 0.069 | |||
END + Adj Tx | 6.043 | 0.717 | 50.911 | 0.098 |
ENI | 8.737 | 1.152 | 66.263 | 0.036 |
END | 2.621 | 0.269 | 25.504 | 0.407 |
Observation | 1 | ? | ? | ? |
HR: Hazard ratio; CI: Confidence interval; END + Adj Tx: Elective neck dissection + adjuvant treatment; ENI: Elective neck irradiation; END: Elective neck dissection.
There were two important observations according to data shown in
Management of the negative neck in patients with HNSCC is a subject of extensive debate. Issue central to the debate usually involves the question whether END would offer a survival advantage over the policy of watchful waiting. When there is an indication for neck treatment, should a patient be treated with END or ENI [
Neck management | VEGF-A and Ki-67 | High risk | Intermediate risk | Low risk | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Dead | Censored | Total | Dead | Censored | Total | Dead | Censored | Total | ||
END + Adj Tx | Both | 2 | 0 | 2 | ? | ? | ? | ? | ? | ? |
Either | 4 | 7 | 11 | ? | ? | ? | ? | ? | ? | |
Neither | 0 | 4 | 4 | ? | ? | ? | ? | ? | ? | |
Total | 6 | 11 | 17 | ? | ? | ? | ? | ? | ? | |
ENI | Both | 0 | 0 | 0 | 3 | 1 | 4 | 2 | 0 | 2 |
Either | 4 | 5 | 9 | 1 | 1 | 2 | 1 | 2 | 3 | |
Neither | 2 | 0 | 2 | 3 | 6 | 9 | 1 | 1 | 2 | |
Total | 6 | 5 | 11 | 7 | 8 | 15 | 4 | 3 | 7 | |
END | Both | 0 | 0 | 0 | 1 | 0 | 1 | ? | ? | ? |
Either | 0 | 3 | 3 | 1 | 2 | 3 | ? | ? | ? | |
Neither | 0 | 5 | 5 | 1 | 9 | 10 | ? | ? | ? | |
Total | 0 | 8 | 8 | 3 | 11 | 14 | ? | ? | ? | |
Observation | Both | ? | ? | ? | ? | ? | ? | 0 | 2 | 2 |
Either | ? | ? | ? | ? | ? | ? | 1 | 4 | 5 | |
Neither | ? | ? | ? | ? | ? | ? | 0 | 9 | 9 | |
Total | ? | ? | ? | ? | ? | ? | 1 | 15 | 16 |
END + Adj Tx: Elective neck dissection + adjuvant treatment; ENI: Elective neck irradiation; END: Elective neck dissection.
Analysis | END + Adj Tx | ENI | END | Observation | |
---|---|---|---|---|---|
END + Adj Tx | Kaplan-Meier survival | ? | 0.308 | 0.099 | 0.052 |
Contingency table | ? | 0.165 | < 0.001 | 0.013 | |
ENI | Kaplan-Meier survival | 0.308 | ? | 0.005 | 0.005 |
Contingency table | 0.165 | ? | 0.002 | 0.025 | |
END | Kaplan-Meier survival | 0.099 | 0.005 | ? | 0.517 |
Contingency table | <0.001 | 0.002 | ? | 0.327 | |
Observation | Kaplan-Meier survival | 0.052 | 0.005 | 0.517 | ? |
Contingency table | 0.013 | 0.025 | 0.327 | ? |
END + Adj Tx: Elective neck dissection + adjuvant treatment; ENI: Elective neck irradiation; END: Elective neck dissection.
At our institute, strategy of neck management has been based on the site and T class of the primary tumor. The decision to treat the neck was made when a risk of subclinical neck disease ≥ 20% [
The aim of this study was to investigate whether the combined expression of VEGF-A and Ki-67 would provide prognostic information useful for treatment selection or adjustment. Previously, coexpression of VEGF-A and Ki-67 was observed to be an aggressive tumor phenotype for the high likelihood of 6.46 to observe lymph node metastases especially in early stage tumor of the oral cavity, oropharynx and hypopharynx [
In our study, 8 out of 11 patients with VEGF-A and Ki-67 coexpression died regardless the type of neck management. This raised a question whether such an expression profile reflected the microinvasiveness of the primary tumor in addition to the presence of the occult neck diseases. The 5-year DSS rate was 34.9% for patients with positive nodes in contrasting to the 11.8% for marker coexpression in the setting of N0 neck. Therefore, marker coexpression in patients with N0 neck might indicate the microinvasiveness of the primary tumor as well as the risk of occult neck disease. Furthermore, among 8 patients who failed, there were only 2 cases receiving the adjuvant treatment of the primary tumor. This led to the postulation that suboptimal treatment of patients with such an aggressive biological feature could be the cause of failure. Henceforth, VEGF-A and Ki-67 coexpression might serve an index in addition to tumor margins in identifying patients who might be benefited from the adjuvant treatment.
On the contrary, there was a question whether neither marker expression would indicate the absence of subclinical neck disease due to a favorable 5-year DSS rate of 81.2% and therefore would be used as an indicator suggesting neck management by wait-and-observe policy. In this subset of 41 patients, 19 were treated by END (with and without adjuvant treatment), 13 by ENI and 9 by observation. If the subclinical neck disease was absence, one could anticipate the nonsignificant treatment outcomes among these different modalities of neck management. But in fact, the odds of being survived for patients treated by END (with and without adjuvant treatment) were 15 times greater than those treated by ENI. The survival benefit for patients with neither marker expression was therefore due to surgical removal of lymph nodes containing nondividing disseminated tumor cells which were radioresistant.
In the use of END or ENI in treating patients of the same risk category, i.e. intermediate to high risks, a better survival was observed for END with a 5-year DSS rate of 85.1% as opposed to 44.7% for ENI. How was this observation compared to other studies? In general, ENI is an option for patients who are poor surgical candidates. Limited studies were conducted to allow the comparison of the effectiveness between END and ENI. In a prospective study using similar clinical criteria, i.e. tumor size and site, in management of cN0 neck for patients with SCC of the oral cavity, the authors reported the 3-year DSS rates of 86% for END and 67% for ENI [
In comparison of treatment outcomes between END and observation which involved patients of different risk categories, i.e. intermediate to high risk for END and low risk for observation, survival equivalent between these neck management modalities should not be misjudged as similarity in treatment effects but rather the equivalence in outcomes of neck management in different clinical contexts, i.e. the effectiveness of END in removal of the subclinical neck diseases and the absence of the occult metastasis in close observation. In spite of the favorable treatment efficacy with END, there is still a concern on the unnecessary neck treatment for approximately 70% of patients who actually do not harbor the metastatic cells in their necks. In indentifying whom to be treated or spared from the neck treatment, we may need a sensitive and reliable method like sentinel node biopsy (SNB) [
Head and neck cancers are heterogeneous in anatomical location and biological behavior. We acknowledge our study limitations for the inclusion of cancer arising from the oral cavity and the larynx and the small number of patients. Further study with large sample size is warranted to ascertain the consistency of the findings so as to gain acceptance of the use of these biomarkers as a complementary tool to the standard clinicopathological criteria in selecting the best treatment for individual patients.
Coexpression of VEGF-A and Ki-67 was a significant prognostic factor independent of modalities of neck management in the setting of N0 neck. Such a pattern of marker expression is a suggestion of an aggressive tumor phenotype implicating the microinvasiveness of the primary tumor in addition to the risk of lymph node metastasis defined previously [
The authors would like to acknowledge the grant support from Mahidol University, Bangkok, Thailand.
VipaBoonkitticharoen,BoonchuKulapaditharom,NoppadolLarbcharoensub,PhurichPraneetvatakul,ThongchaiBhongmakapat, (2015) Prognostic Significance of Vascular Endothelial Growth Factor-A (VEGF-A) and Ki-67 Expression in Head and Neck Cancer Patient with Negative Neck. International Journal of Otolaryngology and Head & Neck Surgery,04,356-368. doi: 10.4236/ijohns.2015.45060