Does angiogenesis have a prognostic value in squamous cell carcinoma of the larynx?

Introduction Angiogenesis marks a tumour′s transition to a systemic disease. It is a useful prognostic and predictive factor; yet, its relationship to prognosis in squamous cell carcinoma of the larynx is controversial. Aim of this study was to evaluate the significance of angiogenesis in laryngeal squamous cell carcinoma. Patients and methods A prospective study was conducted in the Otorhinolaryngology Department of Ain Shams University. Patients with laryngeal cancer, between 2006 and 2012, were enrolled. After surgery, histopathological examination with grading of tumours and immunostaining with CD34 antibody was carried out. Patients were followed up for 2 years and reassessment with immunostaining was carried out for recurrent tumours. Results Eighty cases were included. Results showed that tumours associated with a higher mean microvessel count had a highly significant association with nodal metastasis, recurrence and subsites, with poorer prognosis. Conclusion Results suggest that angiogenesis can be used as a prognostic factor in patients with laryngeal squamous cell carcinoma.


Introduction
Angiogenesis is believed to be a turning point in the development of solid tumours. During the avascular phase, the growth of the tumour is limited by the rate at which nutrients and metabolites diff use across the tumour boundary, and, as a result, are typically only a few millimetres in radius. Following angiogenesis, the tumour has its own vascular network, which provides access to a limitless supply of resources and permits unlimited growth of the tumour. Th e vasculature also provides a route for tumour cells that break free to metastasize. Th us, angiogenesis marks a tumour's transition from a localized lesion to a systemic and potentially fatal disease [1].
Cancer-induced angiogenesis is a result of increased expression of angiogenic factors, such as vascular endothelial growth factor ( VEGF), or decreased expression of antiangiogenic factors, or a combination of both events [2].
Over the last decade, assessment of angiogenesis has emerged as a potentially useful biological prognostic and predictive factor in solid human tumours [3]. Mean microvessel density ( MVD) has been proven to be a powerful and often independent prognostic indicator for many diff erent types of human cancers [4][5][6].
In addition, a signifi cant correlation between the expression of VEGF and prognosis has been described [7,8]. Studies indicate that the levels of angiogenic factors in tissue refl ect the aggressiveness with which tumour cells spread and thus have predictive value in the identifi cation of high-risk patients with poor prognosis. Yet, the results of studies on tumour angiogenesis and the relationship between clinicopathological factors and prognosis in head and neck tumours, particularly in squamous cell carcinoma of the larynx (LSSC), are controversial [8][9][10][11][12][13][14][15].

Introduction
Angiogenesis marks a tumour's transition to a systemic disease. It is a useful prognostic and predictive factor; yet, its relationship to prognosis in squamous cell carcinoma of the larynx is controversial. Aim of this study was to evaluate the signi cance of angiogenesis in laryngeal squamous cell carcinoma.

Patients and methods
Th is was a prospective study conducted at Ain Shams University Hospitals (Otorhinolaryngology and Pathology Departments) between 2006 and 2012 on 80 patients. All patients scheduled for surgical excision of laryngeal cancer were eligible for inclusion in the study. Exclusion criteria included the presence of inoperable or recurrent tumour, previous chemotherapy or radiation of the neck, and presence of end-stage organ disease such as chronic renal failure or liver cell failure. Approval was obtained from the ethical and moral committee of the Faculty of Medicine, Ain Shams University, for the design and protocol of the study, which complied with the Helsinki Declaration of 1975, as revised in 2008, and informed written consent was obtained from all patients before enrolment.
All patients were subjected to extensive workup including history taking, general and otorhinolaryngeal examination, preoperative evaluation including computed tomography scan of the neck and chest, abdominal sonography, direct laryngoscopy for biopsy, and histopathological confi rmation of tumour. TNM staging of the tumour was also carried out.
After defi nitive surgery (partial or total laryngectomy), routine histopathological examination of specimens was carried out using haematoxylin and eosin stains. Slides were examined to confi rm the diagnosis of LSSC and histopathological grading was done. In addition, specimens were immunostained for CD34 antibody using streptavidin-biotin peroxidase. Immunological detection was achieved with the commercially available monoclonal antibody 133, which stains CD34 antigen on endothelial and haematopoietic cells (Ab no. 353M; BioGenex, Biogenex Fremont, CA, USA). Th e super-sensitive antibodies had been optimally diluted by BioGenex and were ready for use without further dilution. Also, horseradish peroxidase kits ( DAB kits) were prepared as described by the manufacturers.
Th e areas of maximal density were identifi ed by scanning with the low power of a light microscope (×100). Th e microvessels were counted in fi ve separate high-power fi elds (´200) [Figs 1 and 2] with the help of a computer-assisted Olympus microscope using Soft Image System Analysis Five (Tokyo, Japan). Th e blood vessels were counted following the criteria of Weidner et al. [16]. A single countable microvessel was defi ned as any brown-stained endothelial cell or group of cells that were clearly separated from other vessels, stroma, or tumour cells without the necessity of a vessel lumen or red blood cell within the lumen. Areas of gross haemorrhage and necrosis were avoided. Branching structures were counted as a single vessel.
Th e mean microvessel count of the fi ve most vascular areas was taken as MVD1, which was expressed as the absolute number of microvessel per 0.74 mm² (×200) fi eld.
All patients were followed up clinically for detection of any recurrence at 3 and 6 months and at 1 and 2 years after laryngectomy by means of a postoperative computed tomography scan of the neck and by direct laryngoscopy for those with partial laryngectomy at 3 months postoperatively; assessment of MVD was carried out in those with recurrent tumour (MVD2).

Statistical analysis
All data were entered and processed usin g SPSS (version 17; SPSS Inc., Chicago, Illinois, USA) using the Mann-Whitney U-test, the Wilcoxon signed-rank test and analysis of variance.
Th e MVD1 for patients with glottic carcinoma ranged from 1 3.25 to 37.6 (mean 28.631 ± 6.539) and that for supraglottic cases ranged fr om 32 to 87.2 (mean 49.6 ± 16.139). Th ere was a highly statistically signifi cant diff erence among subgroups according to the site of cancer (Table 1). After 2 years of follow-up, 16 (20%) patients were seen to have developed recurrence: two (12.5%) patients developed recurrence after 6 months, 12 (75%) patients after 1 year and two (12.5%) patients after 1 year. As regards the site of recurrence, 12 (75%) patients developed in the stoma, two (12.5%) in the lymph nodes and two (12.5%) in the larynx itself (surgery was primarily cordectomy). Th e initial MVD (MVD1) for the 64 patients who did not experience recurrence ranged from 17.2 to 49 (mean 32.972 ± 7.912) and that for recurrent cases ranged from 13.25 to 87.2 (mean 49.956 ± 26.91), with no statistically signifi cant diff erence (Table 5).
On comparing MVD1 with MVD2 (the mean vascular density after recurrence) in the 16 recurrent cases, the following was found: MVD1 ranged from 13.25 to    (Table 6).

Discussion
Th eoretically, angiogenesis should be a poor prognostic factor in neoplasms as it allows the tumour to acquire new blood vessels supplying nutrients to tumour cells; it is also thought to be a way for tumour cells to metastasize, turning a local disease into a systemic one. Yet, confl icting evidence exists about the role of angiogenesis and the importance of MVD as a prognostic factor in head and neck squamous cell carcinoma (HNSCC). According to many reports [13,17,18], high MVD was associated with metastasis and/or a worse clinical outcome. Moreover, VEGF expression was reported to correlate with tumour diff erentiation and stage [8,19]. Also, a correlation with lymph node metastases was shown [8,20]. However, other reports [15,[21][22][23] failed to demonstrate such a relationship.
Th e purpose of this study was to clarify the relationship between the degree of angiogenesis and clinical and prognostic parameters in pat ients with LSCC, which may lead to the application of promising antiangiogenesis therapy to patients with LSCC. Th e degree of angiogenesis was determined by quantifying the per centage of CD34-related antigen immunostaining of vascular endothelial cells found in laryngeal tumour specimens.
We found a statistically signifi cant correlation between MVD and occurrence of regional nodal metastasis, which is considered to be the most important prognostic index in HNSCC. Th is agrees with the theory that tumours with more angiogenesis have greater tendency to metastasize and therefore have a poorer prognosis, which was also observed by other reports [9,24]. In contrast, other studies [12,25] did not show such a statistical correlation; yet, they stated that there was a 'tendency' for an increased degree of angiogenesis in tumours with metastases in regional lymph nodes.
A highly statistically signifi cant correlation was found between MVD and the site of tumour, as supraglottic tumours, known to have a higher incidence of nodal metastasis and poorer prognosis, were found to have a statistically signifi cantly higher MVD compared with glottic tumours, which are known to have better prognosis. Similar results were reported by Do et al. [23]; however, they were not reproducible [11,13].
Further, in our study a positive correlation was found between MVD and pathological grade of the primary tumour, with some studies [8,19,25] confi rming this result and others [15,20,24] failing to demonstrate such a relation. Th e increased MVD in less-diff erentiated tumours in our study suggests that the increase in MVD may be related to loss of diff erentiation. Th is may also be a cause for the greater aggressiveness of less-diff erentiated tumours.
Th e results of our study failed to demonstrate that MVD is a predictor of recurrence in LSSC. Yet, when comparing the initial MVD with MVD after recurrence for the 16 patients who experienced recurrence (MVD2), a statistically highly signifi cant diff erence was found. Th is may be because recurrent tumours are usually more aggressive. Yet, it casts a shadow on the possibility of using MVD as a prognostic factor for recurrence. Th is result was also reported by Hagedorn and Nerlich [11], Pietruszewska et al. [13], and Pignataro et al. [26], although other studies [9,14,27,28] have shown contradictory results.
Th e causes of these contradicting results can be attributed to the fact that direct methods for measuring angiogenic activity in humans are lacking, which is a major diffi culty in studying angiogenesis. Th e relationship between MVD and prognostic parameters in HNSCC could not be completely explained [29].
Although it is diffi cult to measure angiogenesis in  cancers, the determination of MVD (quantifi cation of microvessels stained with immunohistochemical stains in histological sections) is considered to be a reliable indicator [16]. Yet, the measurement of microvasculature in histological sections does not necessarily represent the angiogenic status of the tumour [30,31]. Another reason for these confl icting results is that th e sections analysed may not represent the true state of tumour angioge nesis.
Th e standardization of antibodies and immunohistochemical techniques and the use of automated methods for the determination of vascularity could eliminate some of these problems, but the use of these techniques requires highly specialized equipment [32]. Th e use of a computer-assisted analysis of the microvasculature in tumours seems to be promising, but the wide use of these techniques requires special training and equipment [33]. It is obvious that the debate for the ideal method for microvessel counting still exists. Moreover, antibodies used to highlight blood vessels cannot distinguish between resting and active angiogenic vessels.
In addition, there is no agreement about which types of antibodies should be used in microvessel counting, with diff erent researchers using diff erent antibodies [8,31,33], which may contribute to varied results. Moreover, some studies include patients treated with diff erent therapeutic modalities (surgery or radiation), which can be infl uenced in diff erent ways by tumour vascularization [15].

Conclusion
Our results demonstrate that tumours associated with a higher mean microvessel count are associated with a more aggressive nature, such as those with nodal metastasis, or recurrence, and tumours arising from sites with poorer prognosis. Th is relation was also observed with tumours with a higher T stage, although it was not statistically signifi cant. Th ese results suggest that angiogenic activity can be used as a prognostic factor in patients with LSSC; further, it can direct laryngologists to a more aggressive approach in the treatment and follow-up of suc h tumours.