- Email: [email protected]
Prognostic significance of angiogenesis in surgically treated supraglottic squamous cell carcinomas of the larynx
Acta Otorrinolaringol Esp. 2009;60(4):272-277
Acta Otorrinolaringológica Española www.elsevier.es/otorrino
ORIGINAL ARTICLE
Prognostic significance of angiogenesis in surgically treated supraglottic squamous cell carcinomas of the larynx Juan Pablo Rodrigo,a,* Rubén Cabanillas,a María Dolores Chiara,a Juana García Pedrero,a Aurora Astudillo,b and Carlos Suárez Nietoa a Servicio de Otorrinolaringología, Hospital Universitario Central de Asturias, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Asturias, Spain b Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Asturias, Spain
Received December 17, 2008; accepted December 17, 2008 Available online June 11, 2009
KEYWORDS Squamous cell carcinoma; Supraglottis; Angiogenesis; Prognosis
Abstract Introduction and objectives: It is now generally accepted that angiogenesis is crucial in tumour growth. However, controversy still exists regarding the prognostic significance of angiogenesis in head and neck carcinomas. The aim of this paper is to determine the prognostic significance of angiogenesis in a homogeneously treated group of supraglottic squamous cell carcinomas. Material and methods: Hundred eight patients surgically treated for squamous cell carcinoma of the supraglottic larynx were studied. Angiogenesis was estimated in the primary tumour and in the nodal metastases by determining microvessel density using the “hot spot” method. AntiCD34 antibody was used to stain blood vessels. Results: The mean microvessel density in primary tumours was 72 (34) vessels/mm2 and 58.5 (31.5) vessels/mm2 in nodal metastases. No correlation was found between microvessel density in the primary tumours and the corresponding nodal metastasis (P=.195). No significant differences in microvessel density were observed in relation to clinico-pathological parameters or survival (P=.19). Conclusions: Our results suggest that microvessel density is not a useful prognostic marker in surgically treated supraglottic squamous cell carcinomas. © 2008 Elsevier España, S.L. All rights reserved.
*Corresponding author. E-mail address: [email protected] (J.P. Rodrigo).
0001-6519/$ - see front matter © 2008 Elsevier España, S.L. All rights reserved.
Angiogenesis in supraglottic carcinomas
PALABRAS CLAVE Carcinoma epidermoide; Supraglotis; Angiogénesis; Pronóstico
273
Significado pronóstico de la angiogénesis en los carcinomas epidermoides de laringe supraglótica tratados con cirugía Resumen Introducción y objetivo: La angiogénesis se considera esencial en el crecimiento y la progresión tumorales. Sin embargo, el significado pronóstico de la angiogénesis en los carcinomas de cabeza y cuello es controvertido. El objetivo de este estudio es determinar el significado pronóstico de la angiogénesis en un grupo de carcinomas epidermoides supraglóticos tratados de forma homogénea. Material y métodos: Se estudian 108 pacientes con carcinoma epidermoide de supraglotis tratados quirúrgicamente. La angiogénesis se evaluó en los tumores primarios y en las metástasis ganglionares midiendo la densidad microvascular mediante el método de los “puntos calientes”. El anticuerpo empleado para teñir los vasos sanguíneos fue el anti-CD34. Resultados: La densidad microvascular media en los tumores fue de 72 ± 34 vasos/mm2 y de 58,5 ± 31,5 vasos/mm2 en las metástasis ganglionares. No existía correlación entre la vascularización de los tumores primarios y la de sus metástasis (P = 0,165). No había diferencias significativas en la densidad microvascular en función de los parámetros clínico-patológicos estudiados o de la supervivencia (P = 0,19). Conclusiones: La densidad microvascular no parece ser un marcador pronóstico útil en los carcinomas de laringe supraglótica tratados mediante cirugía. © 2008 Elsevier España, S.L. Todos los derechos reservados.
Introduction
Material and methods
Angiogenesis is currently recognized as a crucial phenomenon in tumour growth and the development of metastasis.1-5 As in other cancers, angiogenesis has been also been implicated in the progression of squamous cell carcinomas of the head and neck.6-10 The general concept is that angiogenesis promotes invasion and metastasis. However, studies on the clinical relevance of angiogenesis in different types of tumours have yielded conflicting results, and these discrepancies have also been found in squamous cell carcinomas of the head and neck. Thus, some studies have described an association between an increased angiogenesis with unfavourable clinical-pathological conditions and a worse prognosis,6,11-17 while others have not found this association.18-24 Although it is difficult to measure angiogenesis in cancers, the determination of microvascular density (quantification of microvessels stained by immunohistochemistry in histological sections) is considered to be a reliable indicator.3 Among the methods to determine microvascular density, the “hot spots” technique is one of the most reproducible.23 One of the possible causes of the discrepancies noted in the medical literature is the inclusion in the study of head and neck tumours of different sublocations, which are known to have different prognoses and different genetic alterations.25 To avoid this problem in this study, we selected a group of patients with tumours in the same location (supraglottic larynx) and with a homogeneous treatment (surgery). The objective is to determine the predictive value of microvascular density in these tumours.
Patients Samples were studied from 108 patients with squamous cell carcinoma of the supraglottic larynx, operated on between 1988 and 1994. All the cases involved primary tumours that had not previously received treatment. The patients were operated on with curative intent and subjected to supraglottic laryngectomy or total laryngectomy plus bilateral lymph node dissection in all cases. Fifty-one patients (47%) received post-operative radiation therapy. This was administered to patients with locally advanced stage (T4) or nodal stage N2-N3. All patients were male, with an average age of 62 years (range, 40-85). All except for one had a history of habitual consumption of tobacco and 94, also of alcohol. Patients were staged according to the classification of the International Union Against Cancer (5th edition). The clinico-pathologic characteristics of the patients are shown in Table 1. Samples of the primary tumour, and also the corresponding lymph node metastases when present, were obtained from the pathology department’s archives.
Immunohistochemical study The tumour samples embedded in paraffin were cut into sections of 4 µm and fixed on siliconized slides (DakoCytomation). The sections were deparaffinized and hydrated in a conventional manner. Antigen retrieval was
274
J.P. Rodrigo et al
Statistical analysis
Table 1 Clinical and pathogenic characteristics of the cases studied Characteristics Number of patients
Microvascular density, mean (SD)
pT classification T1 12 62.2 (28.4) T2 41 74.6 (35.2) T3 39 76.6 (36.5) T4 16 62.5 (29.2) pN classification N0 51 75 (31.2) N1 20 68.6 (36.5) N2 24 72.8 (40.5) N3 13 65.2 (30.5) Stage I 7 67 (32.5) II 23 77.6 (33.5) III 33 71 (32.6) IV 45 71 (34) Histological grade Well 49 80.7 (29.8) differentiated Moderately 38 57.4 (32) differentiated Poorly 21 79 (21) differentiated
Pa
.36
.78
Statistical analysis was performed using version 11.0 of the SPSS programme. To analyze the association existing between microvascular density and clinical-pathological parameters we used the analysis of variance. To establish the correlation between the vascularization of primary tumours and their lymph node, metastases we used the Spearman correlation coefficient. Patient survival was calculated using the Kaplan-Meier method, and differences were analyzed using the log-rank method. For the multivariate study, we used the Cox regression model. Values lower than .05 were considered statistically significant.
Results .84
.003
SD indicates standard deviation. aAnalysis of variance.
performed by heating sections in citrate buffer for 10 min in a pressure cooker. Staining reactions were performed at room temperature in an automated work station (DakoCytomation) in a single session. The samples were 15 min in blocking solution (3% hydrogen peroxide) and then reacted with primary antibody, anti-CD34 clone QBEnd/10 (Novocastra) at a 1:50 dilution. Immunodetection was performed using the Envision system (Envision Plus, Dako) with diaminobenzidine being used as chromogen. The final step was staining with haematoxylin for 1 min. After staining, the sections were dehydrated and mounted with coverslips using a standard medium. Negative controls with omission of primary antibody were included. The preparations were studied at random by 2 of the authors without access to the clinical data. Angiogenesis was evaluated by determining the vascularization of tumours and their metastases using the method proposed by Weidner3; to this end, the number of existing vessels were quantified at a magnification of 200 increases in the 4 tumour areas with the largest number of stained vessels (“hot spots”), determining the number of vessels per mm2. The highest score among the 4 areas quantified was taken as the degree of vascularization (vessels/mm2, microvascular density). For the statistical analysis of survival, the cases were divided according to the median of the degree of vascularization. Primary tumours and metastases were analyzed in different sessions.
The average microvascular density in the 108 tumours studied was 72 vessels/mm2 (standard deviation, 34; range, 14-206), and the median was 68.5 vessels/mm2. In 46 cases it was possible to study the microvascular density in samples from the corresponding lymph node metastases, with an average of 58.5 vessels/mm2 (standard deviation, 31.5 vessels/mm2; range, 23-150). We did not find a significant correlation between microvascular density of primary tumours and their lymph node metastases (P=.165). Figure 1 shows an example of a “hot spot” in a tumour. No significant relationships were found between the microvascular density and the clinical-pathological parameters analyzed (T classification, lymph node metastasis, and stage of disease; Table 1), except for the degree of differentiation, which presented a lower vascularization than moderately differentiated tumours (P=.003; Table 1). Of the 108 patients studied and after 5 years of followup, 60 (56%) were alive and disease-free, 28 (26%) had died because of the tumour, and 20 (18%) had died from other causes. The latter were eliminated from the analysis of
Figure 1 Example of the staining of blood vessels with anti-CD34 antibody in supraglottic epidermoid carcinoma. Haematoxylin, ×200.
Angiogenesis in supraglottic carcinomas
275
Discussion
recurrences. Of the remaining 88 patients, 7 (8%) presented local recurrence, 23 (26%) regional recurrence, and 3 (3.5%) distant metastases. The disease specific survival at 5 years was 70.6% and overall survival was 57%. We found no difference in the incidence of relapses in terms of microvascular density (Table 2). To analyze the impact of microvascular density in the survival of patients, values were dichotomized into high and low degrees of vascularization using the median as the watershed. In the univariate study, factors significantly associated with a worse prognosis were the presence of lymph node metastases (P<.001) and the disease stage (P=.016). There were no significant differences in disease-specific survival according to the degree of vascularization of the tumours (P=.19; Figure 2), even though we were able to see better survival in cases with increased vascularization. In the multivariate study, the only factor significantly associated with disease-specific survival was the presence of lymph node metastasis (relative risk, 6.8; 95% confidence interval, 2.3-19.8; P=.0004; Figure 3).
Angiogenesis is the formation of new blood vessels from the endothelium of pre-existing vessels. This is a complex process that involves multiple steps: remodelling of the extracellular matrix, proliferation and migration of endothelial cells, differentiation into capillaries, anastomosis, and finally formation of the vascular lumen.26 Angiogenesis is considered one of the determinant factors for tumour progression in human cancers, including squamous cell carcinomas of the head and neck.6-10 The most widely used method for assessing angiogenesis in conventional pathology examination is the determination of microvascular density, due to its ease of implementation and its reproducibility. Given the relationship between angiogenesis and tumour progression, various attempts have been made to use microvascular density as a prognostic marker in multiple tumour types,15 generally by associating it with a worse prognosis. However, the predictive value of microvascular density in squamous cell carcinomas of the head and neck is controversial. While some studies associate increased microvascular density with increased tumour aggressiveness and a worse prognosis,6,11-17 others do not find this association.18-24 In this paper we have not been able to find any association between angiogenesis (assessed by measuring microvascular density using the “hot spots” method) and clinical-pathological parameters or prognosis. Moreover, those patients with a higher microvascular density showed better survival, although the differences were not statistically significant. The only association found was a lower microvascular density in moderately differentiated carcinomas, although this association is probably coincidental as the microvascular density values were similar in both well- and poorly-differentiated tumours, which represent the two extremes of histological differentiation. Nor did
Table 2 Relationship between microvascular density and tumour recurrences Recurrences
Number of patients
Microvascular density, mean (SD) Pa
No recurrence Local Regional Distant metastases
55 7 39 16
73.3 (36) 61 (23.4) 72.5 (31.7) 66.7 (18.5)
.83
SD indicates standard deviation. aAnalysis of variance.
1.0 P=.19
0.9 High vascularization
Accumulated survival
0.8 0.7 0.6
Low vascularization
0.5 0.4 0.3 0.2 0.1 0.0 0
20
40
60
80
100
120
140
Months
Figure 2 Disease-specific survival calculated using the Kaplan-Meier method in terms of microvascular density; above the median (high vascularization), and below the median (low vascularization).
276
J.P. Rodrigo et al
1.0
P=.0004
0.9
N0
Accumulated survival
0.8 0.7 0.6 0.5
N1-3
0.4 0.3 0.2 0.1 0.0 0
20
40
60
80
100
120
140
Months
Figure 3 Disease-specific survival calculated using the Kaplan-Meier method according to the presence or absence of lymph node metastases.
we find in this study any correlation between angiogenesis in primary tumours and their corresponding lymph node metastases. The fact of finding a variable angiogenesis in lymph node metastases shows the existence of this phenomenon in their development. But this angiogenesis does not seem to be determined by the characteristics of the primary tumour, but instead by the microenvironment in which tumour cells are found in the lymph node. We found no other studies in the medical literature to determine the relationship between angiogenesis of primary tumours and their corresponding metastases. There are several possible reasons for the discrepancies found in the literature. Tumour location takes pride of place: many studies include tumours with different locations within those generically classified as carcinomas of the head and neck,6,7,10,11,19,20,23 although it is known that each location presents its own clinico-pathologic and prognostic characteristics.25 Also, some studies include patients treated with different therapeutic modalities (surgery or radiation) which can be influenced in different ways by tumour vascularization. Thus, it is more likely that patients treated with radiation therapy are more sensitive to changes in vascularization, since the effectiveness of radiation therapy is highly dependent on the degree of hypoxia in the tissue.27 However, in those treated with surgery, angiogenesis could influence the aggressiveness of the tumour (for example, by favouring invasion or metastasis), but not the response to treatment. To avoid these possible confounding factors in our study, we selected a homogeneous and well balanced group of patients, with carcinomas in the same location (supraglottic larynx) and treated in the same way (partial or total laryngectomy plus bilateral dissection). Given that only patients with disease-free surgical margins were included, it is unlikely that hypoxia or vascularization could impact the effect of post-operative radiation therapy received by
some patients. In studies of patients treated surgically at a single location (tongue), no prognostic value was found for angiogenesis.17,21,22,24 However, continuing with the differences, the prognostic value has been described in other similar studies.12,13 In laryngeal tumours treated surgically, there are also studies that associate microvascular density with the prognosis.14-16 Other possible confounding factors are the type of antibodies used to stain the capillaries and their variability in the immunohistochemical protocols.17 The antibodies used in most studies are antibodies against factor VIII, antigens CD31 and CD34, and more recently against the CD105 antigen (endoglin), which are specifically found in the vascular endothelium. Some studies17 which have employed more than one of these antibodies have not found significant differences in the resulting microvascular density values. However, the main problem is that the most commonly used antibodies to date (factor VIII, CD31 and CD34) do not distinguish between pre-formed vessels and those which are being generated “ex novo.” This problem seems to be solved by using the CD105 marker antigen, which is only expressed in an endothelium which is actively proliferating.28,29 Thus, in the study of Martone et al,29 microvascular density determined through CD105 was associated with a worse prognosis, whereas that determined with CD34 showed no such association. Nevertheless, studies with this antigen are still scarce and there is no consensus on which antibody is the best for this purpose.30 Finally, another problem inherent to studies of angiogenesis is that there is no direct method to determine angiogenic activity in tumours. In fact, although the determination of microvascular density is the most commonly employed method of estimating angiogenesis, it does not necessarily reflect the angiogenic status of a tumour.17 The preferred method for the determination of microvascular density is
Angiogenesis in supraglottic carcinomas
the “hot spots” method,30 which is the one used in most publications. However, this method does not completely eliminate the variability in the choice of the “hot spots” between different observers, which is its main problem. Moreover, in considering a limited number of histological sections, it may occur that the sections analyzed do not represent the true state of tumour angiogenesis. The standardization of antibodies and immunohistochemical techniques and the use of automated methods for the determination of the vascularity could eliminate some of these problems,31 but the use of these techniques requires highly specialized equipment. In conclusion, in our study we found no association between the different clinical and pathological parameters and microvascular density in squamous cell carcinomas of the supraglottic larynx. In the carcinomas of this location treated with surgery, microvascular density does not appear to be associated with the prognosis of the disease.
Funding Paper financed by the FIS Health Research Fund (PI07/0777), the Social Work of Cajastur Savings Bank and the RTICC (Themed Network for Co-operative Cancer Research) (RD06/0020/0034).
References 1. Weidner N. Angiogenesis as a predictor of clinical outcome in cancer patients. Rev Hum Pathol. 2000;31:403-5. 2. Folkman J, Ingber D. Inhibition of angiogenesis. Rev Semin Cancer Biol. 1992;3:89-96. 3. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1-8. 4. Macchiarini P, Fontanini G, Hardin MJ, Squartini F, Angeletti CA. Relation of neovascularisation to metastasis of non-small-cell lung cancer. Lancet. 1992;340:145-56. 5. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol. 1993;143:401-9. 6. Gasparini G, Weidner N, Maluta S, Pozza F, Boracchi P, Mezzetti M, et al. Intratumoral microvessel density and p53 protein: Correlation with metastasis in head-and-neck squamous-cell carcinoma. Int J Cancer. 1993;55:739-44. 7. Hegde PU, Brenski AC, Caldarelli DD, Hutchinson J, Panje WR, Wood NB, et al. Tumor angiogenesis and p53 mutations: Prognosis in head and neck cancer. Arch Otolaryngol Head Neck Surg. 1998;124:80-5. 8. Zhang SC, Miyamoto S, Kamijo T, Hayashi R, Hasebe T, Ishii G, et al. Intratumor microvessel density in biopsy specimens predicts local response of hypopharyngeal cancer to radiotherapy. Jpn J Clin Oncol. 2003;33:613-9. 9. Lukits J, Timar J, Juhasz A, Dome B, Paku S, Repassy G. Progression difference between cancers of the larynx and hypopharynx is not due to tumor size and vascularisation. Otolaryngol Head Neck Surg. 2001;125:18-22. 10. Tae K, El-Naggar AK, Yoo E, Feng L, Lee JJ, Hong WK, et al. Expression of vascular endothelial growth factor and microvessel density in head and neck tumorigenesis. Clin Cancer Res. 2000;6:2821-8. 11. Albo D, Granick MS, Jhala N, Atkinson B, Solomon MP. The relationship of angiogenesis to biological activity in human
277 squamous cell carcinomas of the head and neck. Ann Plast Surg. 1994;32:588-94. 12. Shpitzer T, Chaimoff M, Gal R, Stern Y, Feinmesser R, Segal K. Tumor angiogenesis as a prognostic factor in early oral tongue cancer. Arch Otolaryngol Head Neck Surg. 1996;122:865–8. 13. Alcalde RE, Terakado N, Otsuki K, Matsumura T. Angiogenesis and expression of platelet-derived endothelial cell growth factor in oral squamous cell carcinoma. Oncology. 1997;54:324–8. 14. Murray JD, Carlson GW, McLaughlin K, Pennington M, Lynn PB, DeRose PB, et al. Tumor angiogenesis as a prognostic factor in laryngeal cancer. Am J Surg. 1997;174:523–6. 15. Beatrice F, Cammarota R, Giordano C, Corrado S, Ragona R, Sartoris A, et al. Angiogenesis: prognostic significance in laryngeal cancer. Anticancer Res. 1998;18:4737–40. 16. Kupisz K, Chibowski D, Klatka J, Klonowski S, Stepulak A. Tumor angiogenesis in patients with laryngeal cancer. Eur Arch Otorhinolaryngol. 1999;256:303–5. 17. Kyzas PA, Stefanou D, Batistatou A, Agnantis NJ. Prognostic significance of VEGF immunohistochemical expression and tumor angiogenesis in head and neck squamous cell carcinoma. J Cancer Res Clin Oncol. 2005;131:624–30. 18. Leedy DA, Trune DR, Kronz JD, Weidner N, Cohen JI. Tumor angiogenesis, the p53 antigen, and cervical metastasis in squamous carcinoma of the tongue. Otolaryngol Head Neck Surg. 1994;111:417–22. 19. Dray TG, Hardin NJ, Sofferman RA. Angiogenesis as a prognostic marker in early head and neck cancer. Ann Otol Rhinol Laryngol. 1995;104:724–9. 20. Zatterstrom UK, Brun E, Willen R, Kjellen E, Wennerberg J. Tumor angiogenesis and prognosis in squamous cell carcinoma of the head and neck. Head Neck. 1995;17:312–8. 21. Gleich LL, Biddinger PW, Pavelic ZP, Gluckman JL. Tumor angiogenesis in T1 oral cavity squamous cell carcinoma: Role in predicting tumor aggressiveness. Head Neck. 1996;18:343–6. 22. Gleich LL, Biddinger PW, Duperier FD, Gluckman JL. Tumor angiogenesis as a prognostic indicator in T2–T4 oral cavity squamous cell carcinoma: A clinical–pathologic correlation. Head Neck. 1997;19:276–80. 23. Tse GM, Chan AW, Yu KH, King AD, Wong KT, Chen GG, et al. Strong immunohistochemical expression of vascular endothelial growth factor predicts overall survival in head and neck squamous cell carcinoma. Ann Surg Oncol. 2007;14:3558–65. 24. Fernández MM, García-Rozado A, Parente PL. ¿Es la densidad microvascular un factor pronóstico independiente en el carcinoma epidermoide de lengua? Acta Otorrinolaringol Esp. 2007;58:341–6. 25. Rodrigo JP, Suárez C, González MV, Lazo PS, Ramos S, Coto E, et al. Variability of genetic alterations in different sites of head and neck cancer. Laryngoscope. 2001;111:1297–301. 26. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31. 27. Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol. 1996; 41:31–9. 28. Schimming R, Marme D. Endoglin (CD105) expression in squamous cell carcinoma of the oral cavity. Head Neck. 2002;24:151–6. 29. Martone T, Rosso P, Albera R, Migliaretti G, Fraire F, Pignataro L, et al. Prognostic relevance of CD105+ microvessel density in HNSCC patient outcome. Oral Oncol. 2005;41:147–55. 30. Vermeulen PB, Gasparini G, Fox SB, Colpaert C, Marson LP, Gion M. Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours. Eur J Cancer. 2002;38:1564–79. 31. Hannen EJ, van der Laak JA, Manni JJ, Freihofer HP, Slootweg PJ, Koole R, et al. Computer assisted analysis of the microvasculature in metastasized and nonmetastasized squamous cell carcinomas of the tongue. Head Neck. 2002;24: 643–50.
Acta Otorrinolaringológica Española www.elsevier.es/otorrino
ORIGINAL ARTICLE
Prognostic significance of angiogenesis in surgically treated supraglottic squamous cell carcinomas of the larynx Juan Pablo Rodrigo,a,* Rubén Cabanillas,a María Dolores Chiara,a Juana García Pedrero,a Aurora Astudillo,b and Carlos Suárez Nietoa a Servicio de Otorrinolaringología, Hospital Universitario Central de Asturias, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Asturias, Spain b Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Asturias, Spain
Received December 17, 2008; accepted December 17, 2008 Available online June 11, 2009
KEYWORDS Squamous cell carcinoma; Supraglottis; Angiogenesis; Prognosis
Abstract Introduction and objectives: It is now generally accepted that angiogenesis is crucial in tumour growth. However, controversy still exists regarding the prognostic significance of angiogenesis in head and neck carcinomas. The aim of this paper is to determine the prognostic significance of angiogenesis in a homogeneously treated group of supraglottic squamous cell carcinomas. Material and methods: Hundred eight patients surgically treated for squamous cell carcinoma of the supraglottic larynx were studied. Angiogenesis was estimated in the primary tumour and in the nodal metastases by determining microvessel density using the “hot spot” method. AntiCD34 antibody was used to stain blood vessels. Results: The mean microvessel density in primary tumours was 72 (34) vessels/mm2 and 58.5 (31.5) vessels/mm2 in nodal metastases. No correlation was found between microvessel density in the primary tumours and the corresponding nodal metastasis (P=.195). No significant differences in microvessel density were observed in relation to clinico-pathological parameters or survival (P=.19). Conclusions: Our results suggest that microvessel density is not a useful prognostic marker in surgically treated supraglottic squamous cell carcinomas. © 2008 Elsevier España, S.L. All rights reserved.
*Corresponding author. E-mail address: [email protected] (J.P. Rodrigo).
0001-6519/$ - see front matter © 2008 Elsevier España, S.L. All rights reserved.
Angiogenesis in supraglottic carcinomas
PALABRAS CLAVE Carcinoma epidermoide; Supraglotis; Angiogénesis; Pronóstico
273
Significado pronóstico de la angiogénesis en los carcinomas epidermoides de laringe supraglótica tratados con cirugía Resumen Introducción y objetivo: La angiogénesis se considera esencial en el crecimiento y la progresión tumorales. Sin embargo, el significado pronóstico de la angiogénesis en los carcinomas de cabeza y cuello es controvertido. El objetivo de este estudio es determinar el significado pronóstico de la angiogénesis en un grupo de carcinomas epidermoides supraglóticos tratados de forma homogénea. Material y métodos: Se estudian 108 pacientes con carcinoma epidermoide de supraglotis tratados quirúrgicamente. La angiogénesis se evaluó en los tumores primarios y en las metástasis ganglionares midiendo la densidad microvascular mediante el método de los “puntos calientes”. El anticuerpo empleado para teñir los vasos sanguíneos fue el anti-CD34. Resultados: La densidad microvascular media en los tumores fue de 72 ± 34 vasos/mm2 y de 58,5 ± 31,5 vasos/mm2 en las metástasis ganglionares. No existía correlación entre la vascularización de los tumores primarios y la de sus metástasis (P = 0,165). No había diferencias significativas en la densidad microvascular en función de los parámetros clínico-patológicos estudiados o de la supervivencia (P = 0,19). Conclusiones: La densidad microvascular no parece ser un marcador pronóstico útil en los carcinomas de laringe supraglótica tratados mediante cirugía. © 2008 Elsevier España, S.L. Todos los derechos reservados.
Introduction
Material and methods
Angiogenesis is currently recognized as a crucial phenomenon in tumour growth and the development of metastasis.1-5 As in other cancers, angiogenesis has been also been implicated in the progression of squamous cell carcinomas of the head and neck.6-10 The general concept is that angiogenesis promotes invasion and metastasis. However, studies on the clinical relevance of angiogenesis in different types of tumours have yielded conflicting results, and these discrepancies have also been found in squamous cell carcinomas of the head and neck. Thus, some studies have described an association between an increased angiogenesis with unfavourable clinical-pathological conditions and a worse prognosis,6,11-17 while others have not found this association.18-24 Although it is difficult to measure angiogenesis in cancers, the determination of microvascular density (quantification of microvessels stained by immunohistochemistry in histological sections) is considered to be a reliable indicator.3 Among the methods to determine microvascular density, the “hot spots” technique is one of the most reproducible.23 One of the possible causes of the discrepancies noted in the medical literature is the inclusion in the study of head and neck tumours of different sublocations, which are known to have different prognoses and different genetic alterations.25 To avoid this problem in this study, we selected a group of patients with tumours in the same location (supraglottic larynx) and with a homogeneous treatment (surgery). The objective is to determine the predictive value of microvascular density in these tumours.
Patients Samples were studied from 108 patients with squamous cell carcinoma of the supraglottic larynx, operated on between 1988 and 1994. All the cases involved primary tumours that had not previously received treatment. The patients were operated on with curative intent and subjected to supraglottic laryngectomy or total laryngectomy plus bilateral lymph node dissection in all cases. Fifty-one patients (47%) received post-operative radiation therapy. This was administered to patients with locally advanced stage (T4) or nodal stage N2-N3. All patients were male, with an average age of 62 years (range, 40-85). All except for one had a history of habitual consumption of tobacco and 94, also of alcohol. Patients were staged according to the classification of the International Union Against Cancer (5th edition). The clinico-pathologic characteristics of the patients are shown in Table 1. Samples of the primary tumour, and also the corresponding lymph node metastases when present, were obtained from the pathology department’s archives.
Immunohistochemical study The tumour samples embedded in paraffin were cut into sections of 4 µm and fixed on siliconized slides (DakoCytomation). The sections were deparaffinized and hydrated in a conventional manner. Antigen retrieval was
274
J.P. Rodrigo et al
Statistical analysis
Table 1 Clinical and pathogenic characteristics of the cases studied Characteristics Number of patients
Microvascular density, mean (SD)
pT classification T1 12 62.2 (28.4) T2 41 74.6 (35.2) T3 39 76.6 (36.5) T4 16 62.5 (29.2) pN classification N0 51 75 (31.2) N1 20 68.6 (36.5) N2 24 72.8 (40.5) N3 13 65.2 (30.5) Stage I 7 67 (32.5) II 23 77.6 (33.5) III 33 71 (32.6) IV 45 71 (34) Histological grade Well 49 80.7 (29.8) differentiated Moderately 38 57.4 (32) differentiated Poorly 21 79 (21) differentiated
Pa
.36
.78
Statistical analysis was performed using version 11.0 of the SPSS programme. To analyze the association existing between microvascular density and clinical-pathological parameters we used the analysis of variance. To establish the correlation between the vascularization of primary tumours and their lymph node, metastases we used the Spearman correlation coefficient. Patient survival was calculated using the Kaplan-Meier method, and differences were analyzed using the log-rank method. For the multivariate study, we used the Cox regression model. Values lower than .05 were considered statistically significant.
Results .84
.003
SD indicates standard deviation. aAnalysis of variance.
performed by heating sections in citrate buffer for 10 min in a pressure cooker. Staining reactions were performed at room temperature in an automated work station (DakoCytomation) in a single session. The samples were 15 min in blocking solution (3% hydrogen peroxide) and then reacted with primary antibody, anti-CD34 clone QBEnd/10 (Novocastra) at a 1:50 dilution. Immunodetection was performed using the Envision system (Envision Plus, Dako) with diaminobenzidine being used as chromogen. The final step was staining with haematoxylin for 1 min. After staining, the sections were dehydrated and mounted with coverslips using a standard medium. Negative controls with omission of primary antibody were included. The preparations were studied at random by 2 of the authors without access to the clinical data. Angiogenesis was evaluated by determining the vascularization of tumours and their metastases using the method proposed by Weidner3; to this end, the number of existing vessels were quantified at a magnification of 200 increases in the 4 tumour areas with the largest number of stained vessels (“hot spots”), determining the number of vessels per mm2. The highest score among the 4 areas quantified was taken as the degree of vascularization (vessels/mm2, microvascular density). For the statistical analysis of survival, the cases were divided according to the median of the degree of vascularization. Primary tumours and metastases were analyzed in different sessions.
The average microvascular density in the 108 tumours studied was 72 vessels/mm2 (standard deviation, 34; range, 14-206), and the median was 68.5 vessels/mm2. In 46 cases it was possible to study the microvascular density in samples from the corresponding lymph node metastases, with an average of 58.5 vessels/mm2 (standard deviation, 31.5 vessels/mm2; range, 23-150). We did not find a significant correlation between microvascular density of primary tumours and their lymph node metastases (P=.165). Figure 1 shows an example of a “hot spot” in a tumour. No significant relationships were found between the microvascular density and the clinical-pathological parameters analyzed (T classification, lymph node metastasis, and stage of disease; Table 1), except for the degree of differentiation, which presented a lower vascularization than moderately differentiated tumours (P=.003; Table 1). Of the 108 patients studied and after 5 years of followup, 60 (56%) were alive and disease-free, 28 (26%) had died because of the tumour, and 20 (18%) had died from other causes. The latter were eliminated from the analysis of
Figure 1 Example of the staining of blood vessels with anti-CD34 antibody in supraglottic epidermoid carcinoma. Haematoxylin, ×200.
Angiogenesis in supraglottic carcinomas
275
Discussion
recurrences. Of the remaining 88 patients, 7 (8%) presented local recurrence, 23 (26%) regional recurrence, and 3 (3.5%) distant metastases. The disease specific survival at 5 years was 70.6% and overall survival was 57%. We found no difference in the incidence of relapses in terms of microvascular density (Table 2). To analyze the impact of microvascular density in the survival of patients, values were dichotomized into high and low degrees of vascularization using the median as the watershed. In the univariate study, factors significantly associated with a worse prognosis were the presence of lymph node metastases (P<.001) and the disease stage (P=.016). There were no significant differences in disease-specific survival according to the degree of vascularization of the tumours (P=.19; Figure 2), even though we were able to see better survival in cases with increased vascularization. In the multivariate study, the only factor significantly associated with disease-specific survival was the presence of lymph node metastasis (relative risk, 6.8; 95% confidence interval, 2.3-19.8; P=.0004; Figure 3).
Angiogenesis is the formation of new blood vessels from the endothelium of pre-existing vessels. This is a complex process that involves multiple steps: remodelling of the extracellular matrix, proliferation and migration of endothelial cells, differentiation into capillaries, anastomosis, and finally formation of the vascular lumen.26 Angiogenesis is considered one of the determinant factors for tumour progression in human cancers, including squamous cell carcinomas of the head and neck.6-10 The most widely used method for assessing angiogenesis in conventional pathology examination is the determination of microvascular density, due to its ease of implementation and its reproducibility. Given the relationship between angiogenesis and tumour progression, various attempts have been made to use microvascular density as a prognostic marker in multiple tumour types,15 generally by associating it with a worse prognosis. However, the predictive value of microvascular density in squamous cell carcinomas of the head and neck is controversial. While some studies associate increased microvascular density with increased tumour aggressiveness and a worse prognosis,6,11-17 others do not find this association.18-24 In this paper we have not been able to find any association between angiogenesis (assessed by measuring microvascular density using the “hot spots” method) and clinical-pathological parameters or prognosis. Moreover, those patients with a higher microvascular density showed better survival, although the differences were not statistically significant. The only association found was a lower microvascular density in moderately differentiated carcinomas, although this association is probably coincidental as the microvascular density values were similar in both well- and poorly-differentiated tumours, which represent the two extremes of histological differentiation. Nor did
Table 2 Relationship between microvascular density and tumour recurrences Recurrences
Number of patients
Microvascular density, mean (SD) Pa
No recurrence Local Regional Distant metastases
55 7 39 16
73.3 (36) 61 (23.4) 72.5 (31.7) 66.7 (18.5)
.83
SD indicates standard deviation. aAnalysis of variance.
1.0 P=.19
0.9 High vascularization
Accumulated survival
0.8 0.7 0.6
Low vascularization
0.5 0.4 0.3 0.2 0.1 0.0 0
20
40
60
80
100
120
140
Months
Figure 2 Disease-specific survival calculated using the Kaplan-Meier method in terms of microvascular density; above the median (high vascularization), and below the median (low vascularization).
276
J.P. Rodrigo et al
1.0
P=.0004
0.9
N0
Accumulated survival
0.8 0.7 0.6 0.5
N1-3
0.4 0.3 0.2 0.1 0.0 0
20
40
60
80
100
120
140
Months
Figure 3 Disease-specific survival calculated using the Kaplan-Meier method according to the presence or absence of lymph node metastases.
we find in this study any correlation between angiogenesis in primary tumours and their corresponding lymph node metastases. The fact of finding a variable angiogenesis in lymph node metastases shows the existence of this phenomenon in their development. But this angiogenesis does not seem to be determined by the characteristics of the primary tumour, but instead by the microenvironment in which tumour cells are found in the lymph node. We found no other studies in the medical literature to determine the relationship between angiogenesis of primary tumours and their corresponding metastases. There are several possible reasons for the discrepancies found in the literature. Tumour location takes pride of place: many studies include tumours with different locations within those generically classified as carcinomas of the head and neck,6,7,10,11,19,20,23 although it is known that each location presents its own clinico-pathologic and prognostic characteristics.25 Also, some studies include patients treated with different therapeutic modalities (surgery or radiation) which can be influenced in different ways by tumour vascularization. Thus, it is more likely that patients treated with radiation therapy are more sensitive to changes in vascularization, since the effectiveness of radiation therapy is highly dependent on the degree of hypoxia in the tissue.27 However, in those treated with surgery, angiogenesis could influence the aggressiveness of the tumour (for example, by favouring invasion or metastasis), but not the response to treatment. To avoid these possible confounding factors in our study, we selected a homogeneous and well balanced group of patients, with carcinomas in the same location (supraglottic larynx) and treated in the same way (partial or total laryngectomy plus bilateral dissection). Given that only patients with disease-free surgical margins were included, it is unlikely that hypoxia or vascularization could impact the effect of post-operative radiation therapy received by
some patients. In studies of patients treated surgically at a single location (tongue), no prognostic value was found for angiogenesis.17,21,22,24 However, continuing with the differences, the prognostic value has been described in other similar studies.12,13 In laryngeal tumours treated surgically, there are also studies that associate microvascular density with the prognosis.14-16 Other possible confounding factors are the type of antibodies used to stain the capillaries and their variability in the immunohistochemical protocols.17 The antibodies used in most studies are antibodies against factor VIII, antigens CD31 and CD34, and more recently against the CD105 antigen (endoglin), which are specifically found in the vascular endothelium. Some studies17 which have employed more than one of these antibodies have not found significant differences in the resulting microvascular density values. However, the main problem is that the most commonly used antibodies to date (factor VIII, CD31 and CD34) do not distinguish between pre-formed vessels and those which are being generated “ex novo.” This problem seems to be solved by using the CD105 marker antigen, which is only expressed in an endothelium which is actively proliferating.28,29 Thus, in the study of Martone et al,29 microvascular density determined through CD105 was associated with a worse prognosis, whereas that determined with CD34 showed no such association. Nevertheless, studies with this antigen are still scarce and there is no consensus on which antibody is the best for this purpose.30 Finally, another problem inherent to studies of angiogenesis is that there is no direct method to determine angiogenic activity in tumours. In fact, although the determination of microvascular density is the most commonly employed method of estimating angiogenesis, it does not necessarily reflect the angiogenic status of a tumour.17 The preferred method for the determination of microvascular density is
Angiogenesis in supraglottic carcinomas
the “hot spots” method,30 which is the one used in most publications. However, this method does not completely eliminate the variability in the choice of the “hot spots” between different observers, which is its main problem. Moreover, in considering a limited number of histological sections, it may occur that the sections analyzed do not represent the true state of tumour angiogenesis. The standardization of antibodies and immunohistochemical techniques and the use of automated methods for the determination of the vascularity could eliminate some of these problems,31 but the use of these techniques requires highly specialized equipment. In conclusion, in our study we found no association between the different clinical and pathological parameters and microvascular density in squamous cell carcinomas of the supraglottic larynx. In the carcinomas of this location treated with surgery, microvascular density does not appear to be associated with the prognosis of the disease.
Funding Paper financed by the FIS Health Research Fund (PI07/0777), the Social Work of Cajastur Savings Bank and the RTICC (Themed Network for Co-operative Cancer Research) (RD06/0020/0034).
References 1. Weidner N. Angiogenesis as a predictor of clinical outcome in cancer patients. Rev Hum Pathol. 2000;31:403-5. 2. Folkman J, Ingber D. Inhibition of angiogenesis. Rev Semin Cancer Biol. 1992;3:89-96. 3. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1-8. 4. Macchiarini P, Fontanini G, Hardin MJ, Squartini F, Angeletti CA. Relation of neovascularisation to metastasis of non-small-cell lung cancer. Lancet. 1992;340:145-56. 5. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol. 1993;143:401-9. 6. Gasparini G, Weidner N, Maluta S, Pozza F, Boracchi P, Mezzetti M, et al. Intratumoral microvessel density and p53 protein: Correlation with metastasis in head-and-neck squamous-cell carcinoma. Int J Cancer. 1993;55:739-44. 7. Hegde PU, Brenski AC, Caldarelli DD, Hutchinson J, Panje WR, Wood NB, et al. Tumor angiogenesis and p53 mutations: Prognosis in head and neck cancer. Arch Otolaryngol Head Neck Surg. 1998;124:80-5. 8. Zhang SC, Miyamoto S, Kamijo T, Hayashi R, Hasebe T, Ishii G, et al. Intratumor microvessel density in biopsy specimens predicts local response of hypopharyngeal cancer to radiotherapy. Jpn J Clin Oncol. 2003;33:613-9. 9. Lukits J, Timar J, Juhasz A, Dome B, Paku S, Repassy G. Progression difference between cancers of the larynx and hypopharynx is not due to tumor size and vascularisation. Otolaryngol Head Neck Surg. 2001;125:18-22. 10. Tae K, El-Naggar AK, Yoo E, Feng L, Lee JJ, Hong WK, et al. Expression of vascular endothelial growth factor and microvessel density in head and neck tumorigenesis. Clin Cancer Res. 2000;6:2821-8. 11. Albo D, Granick MS, Jhala N, Atkinson B, Solomon MP. The relationship of angiogenesis to biological activity in human
277 squamous cell carcinomas of the head and neck. Ann Plast Surg. 1994;32:588-94. 12. Shpitzer T, Chaimoff M, Gal R, Stern Y, Feinmesser R, Segal K. Tumor angiogenesis as a prognostic factor in early oral tongue cancer. Arch Otolaryngol Head Neck Surg. 1996;122:865–8. 13. Alcalde RE, Terakado N, Otsuki K, Matsumura T. Angiogenesis and expression of platelet-derived endothelial cell growth factor in oral squamous cell carcinoma. Oncology. 1997;54:324–8. 14. Murray JD, Carlson GW, McLaughlin K, Pennington M, Lynn PB, DeRose PB, et al. Tumor angiogenesis as a prognostic factor in laryngeal cancer. Am J Surg. 1997;174:523–6. 15. Beatrice F, Cammarota R, Giordano C, Corrado S, Ragona R, Sartoris A, et al. Angiogenesis: prognostic significance in laryngeal cancer. Anticancer Res. 1998;18:4737–40. 16. Kupisz K, Chibowski D, Klatka J, Klonowski S, Stepulak A. Tumor angiogenesis in patients with laryngeal cancer. Eur Arch Otorhinolaryngol. 1999;256:303–5. 17. Kyzas PA, Stefanou D, Batistatou A, Agnantis NJ. Prognostic significance of VEGF immunohistochemical expression and tumor angiogenesis in head and neck squamous cell carcinoma. J Cancer Res Clin Oncol. 2005;131:624–30. 18. Leedy DA, Trune DR, Kronz JD, Weidner N, Cohen JI. Tumor angiogenesis, the p53 antigen, and cervical metastasis in squamous carcinoma of the tongue. Otolaryngol Head Neck Surg. 1994;111:417–22. 19. Dray TG, Hardin NJ, Sofferman RA. Angiogenesis as a prognostic marker in early head and neck cancer. Ann Otol Rhinol Laryngol. 1995;104:724–9. 20. Zatterstrom UK, Brun E, Willen R, Kjellen E, Wennerberg J. Tumor angiogenesis and prognosis in squamous cell carcinoma of the head and neck. Head Neck. 1995;17:312–8. 21. Gleich LL, Biddinger PW, Pavelic ZP, Gluckman JL. Tumor angiogenesis in T1 oral cavity squamous cell carcinoma: Role in predicting tumor aggressiveness. Head Neck. 1996;18:343–6. 22. Gleich LL, Biddinger PW, Duperier FD, Gluckman JL. Tumor angiogenesis as a prognostic indicator in T2–T4 oral cavity squamous cell carcinoma: A clinical–pathologic correlation. Head Neck. 1997;19:276–80. 23. Tse GM, Chan AW, Yu KH, King AD, Wong KT, Chen GG, et al. Strong immunohistochemical expression of vascular endothelial growth factor predicts overall survival in head and neck squamous cell carcinoma. Ann Surg Oncol. 2007;14:3558–65. 24. Fernández MM, García-Rozado A, Parente PL. ¿Es la densidad microvascular un factor pronóstico independiente en el carcinoma epidermoide de lengua? Acta Otorrinolaringol Esp. 2007;58:341–6. 25. Rodrigo JP, Suárez C, González MV, Lazo PS, Ramos S, Coto E, et al. Variability of genetic alterations in different sites of head and neck cancer. Laryngoscope. 2001;111:1297–301. 26. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31. 27. Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol. 1996; 41:31–9. 28. Schimming R, Marme D. Endoglin (CD105) expression in squamous cell carcinoma of the oral cavity. Head Neck. 2002;24:151–6. 29. Martone T, Rosso P, Albera R, Migliaretti G, Fraire F, Pignataro L, et al. Prognostic relevance of CD105+ microvessel density in HNSCC patient outcome. Oral Oncol. 2005;41:147–55. 30. Vermeulen PB, Gasparini G, Fox SB, Colpaert C, Marson LP, Gion M. Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours. Eur J Cancer. 2002;38:1564–79. 31. Hannen EJ, van der Laak JA, Manni JJ, Freihofer HP, Slootweg PJ, Koole R, et al. Computer assisted analysis of the microvasculature in metastasized and nonmetastasized squamous cell carcinomas of the tongue. Head Neck. 2002;24: 643–50.