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The quantification of angiogenesis in relation to metastasis in oral cancer: a review
Int. J. Oral Maxillofac. Surg. 2004; 33: 2–7 doi:10.1054/ijom.2003.0433, available online at http://www.sciencedirect.com
Invited Review Paper Head and Neck Oncology
The quantification of angiogenesis in relation to metastasis in oral cancer: a review
E. J. M. Hannen1, D. Riediger2 1
Department of Oral and Maxillofacial Surgery, Catharina-Hospital Eindhoven, The Netherlands; 2Department of Cranio Maxillofacial Surgery, Head and Neck Surgery, University Hospital Maastricht, The Netherlands
E. J. M. Hannen, D. Riediger: The quantification of angiogenesis in relation to metastasis in oral cancer: a review. Int. J. Oral Maxillofac. Surg. 2004; 33: 2–7. 2003 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Tumours need vessels to grow into clinically significant dimensions, and to metastasize. Varying results have been reported for the relation between angiogenic activity in oral carcinomas, and the occurrence of metastasis. Quantification of microvessels in tissue sections is mostly used to assess angiogenesis, but appears subject to biases as reflected in contradicting reports. Especially the choice of immunohistochemical staining technique appears pivotal. Although microvessel density measurements (MVD) appear promising for other tumour types, in oral carcinomas no unambiguous relation could be established. Furthermore, MVD assessment appears more elaborate than expected. New, more time efficient techniques may replace MVD as we know it.
Introduction Solid tumours are dependent on vascular supply to grow beyond spheres of about 0.5–2 mm in diameter12. For this, they use existing vessels, in a process called co-option24, or they induce growth of newly sprouted vessels, i.e., angiogenesis8. Tumours with superior blood supply are thought to have a growth advantage over poorly vascularized. However, not only are bloodvessels necessary for growth, they also provide the route for possible metastasis. Here too, well-vascularized tumours are thought to metastasize more easily because of facilitated access to intravascular transportation. Ever since Folkman and Weidner postulated such56, many studies addressed this issue 0901-5027/04/010002+06 $30.00/0
in an effort to prove such a relation, and for some tumours there is a body of evidence suggestive in that direction55. But for head and neck carcinoma, varying results have been reported2,11,15,16, 17,18,20,21,23,28,30,33,34,37,38,40,47,48,49,58,61 . As a measure of angiogenic activity, most studies count the number of microvessels in tissue sections, expressed in the microvessel density (MVD)53. This technique was designated an easy prognosticator for clinical behaviour for a number of tumours54,55, but the assay proved more elaborate than expected10,14,53. In oral carcinoma too, no indisputable relation between high MVD and metastasis was found20. This paper summarizes the studies in the field of vascular quantification in relation to metastasis for oral cancer and puts
Key words: neoplasm blood supply; neovascularization; head and neck neoplasms; squamous cell carcinoma; oral carcinoma; review literature; diagnostic techniques and procedures; patient care planning. Accepted for publication 16 April 2003
results into perspective.
clinical
and
biological
Technical considerations MVD assessment was introduced by W56, and the technique basically implies counting of routine immunohistochemically stained vessel wall profiles in tissue sections of tumours25. In its original description, first at low magnification, a domain of high staining intensity in a section was identified, wherein MVD was subsequently determined at high power magnification. Theoretically, this approach introduces two biases. First, by selecting a section from the whole tumour resection specimen10, and second, by selecting a subjectively defined hot spot of angiogenic activity51.
2003 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
The quantification of angiogenesis in relation to metastasis in oral cancer Table 1. Antibodies in use for microvessel visualization in immunohistochemically stained tissue sections Antibody against CD31 (PECAM) CD34 Von Willebrand factor (F VIII-related antigen) CD105 Collagen IV Pal-E VEGF-receptors FLT-4 (VEGFR-3) LYVE-1
Epitope Cell surface antigen expressed by platelets, endothelial cells, granulocytes, some lymphocytes Transmembrane glycoprotein on vascular endothelial cells, haematopoietic precursor cells Reacts with Von Willebrand Factor in endothelial cells TGF- binding protein on endothelial cells Collagen type IV in vessel basal membrane Not known; expression exclusively on blood vessels VEGF-receptors in endothelial cells VEGF-receptor for VEGF-C, predominantly present on lymphatics Hyaluronan receptor on lymphatic endothelium
The carry of these biases apparently varies among tumour types, as some tumours prove robust enough, but for oral carcinomas there is room for doubt20. Limiting the area under investigation to be assessed, however is a practical advantage, improving time efficiency. A range of antibodies for the staining of vessel walls is available (Table 1), most of which are aimed at, but not exclusively selective for, epitopes on endothelial cells. Especially CD31, often advocated as the antibody of choice for many tumours53, is known to cross react with plasma cells, abundantly present in oral carcinomas, i.e., false positive staining may occur. Although no extensive trial comparing a broad panel of antibodies is presently available, most suited for oral specimens is CD3419,20,45. The sensitivity of staining, i.e., whether all endothelial cells are recognizably and completely stained, depends among others on the specimen fixation technique, but to a greater extent on the immunohistochemical staining procedure19. Certainly if an automated quantification technique is to be employed (vide infra), enhancement of staining is usually necessary19. Most used antibodies react both with blood vessels as well as lymph vessels (Table 1). The visual identification of the vessel transsections in the tissue section through the microscope may appear trivial if good staining quality is accomplished, but tumour vessels are mostly of aberrant morphology, tortuous, without clear lumen and with large gaps between endothelial cells. This leaves room for discussion, whether a single endothelial cell represents a true vessel wall, or not, and whether it represents a vessel suitable for transportation of malignant cells (Fig. 1). W et al.53,56 proposed to include all endothelial cells,
regardless of the above issues, and most studies comply. However, results from studies aimed at perfusion rate to quantify a tumour’s vascular dependence7,52 may deviate from MVD studies for this reason. All these considerations taken together make MVD assessment a time consuming and laborious effort. The introduction of image analysis14,19,51 improves time management, because multiple, complete tissue sections can be measured in an automated, self-
3
controlled process, reducing sampling error. Furthermore, stained objects are recognized identically in repeated measurements, eliminating intra and inter observer errors. Image analysis, however, requires excellent tissue and staining qualities: frozen sections are unsuited because of insufficient morphology. Besides, image analysis is not ubiquitously available, and requires the assistance of an image analysis expert. Overview of the literature Studies exclusively studying oral carcinoma are very few. To date, 15 studies have quantitated MVD in head and neck squamous cell carcinoma (HNSCC); some of them have studied its relation to metastasis. Despite efforts to introduce standard procedures, techniques differ among studies. Furthermore, not all studies describe all technical details satisfactorily, as to which vessels were included, and as to the definition of metastasis. Data are reported as provided in the respective studies. To facilitate comparison for this review, the results have been recalculated into number of vessels per mm2 in Table 2. Large differences between
Fig. 1. Tissue section of a tongue carcinoma, stained for CD34, with hematoxylin counterstaining19. Red profiles represent endothelial structures. Does the black arrow point at one or two vessels? Is the red stain (green arrow) a vessel, or not?
4
Hannen and Riediger
Table 2. Results reported in HNSCC microvessel density studies, recalculated into counts per mm2 Standard Deviation (SD, where available). Values for the selected hot spot and mean overall values depending on available data
Author A2 G15 G16 G17 G17 G18 H20 H21 H21 L30 M33 M34 P37 P38 S47 T49
Tissue Oral scc HNSCC HNSCC T2–4 tongue+FOM‡ scc T2–4 tongue+FOM‡ scc T1 tongue+FOM‡ scc T1–4 tongue scc HNSCC Tongue¶ scc T1–2 tongue scc Oral scc Tongue scc Oral scc Oral scc T1 tongue scc Lip scc
Antibody F VIII CD31 CD31 F VIII CD31 CD31 CD34 F VIII or CD31 F VIII or CD31 F VIII F VIII CD31 F VIII CD31 F VIII F VIII
Mean valueSD for vessels per mm2 in hot spot Lymphnode Lymphnode+
Mean valueSD for vessels per mm2 overall Lymphnode Lymphnode+ 22.76.8
29a
71a
97.446.6 168.0 206.5 157.7 178.5 193.0 192.0 264.772.1 194.456.4 38.3 36.6
107.862.2
82.349.5
75.0/107.0*
72.9/101.0*
14271 18.3§ 14853 11624 50.013.7† 65.6/137.8*
16856 103.440.7† 59.4/118.9*
a Median; ‡floor of the mouth; *depending on magnification (200/400, respectively); †per 250 magnification field; §per 200 magnification field; ¶two cases from HN group21.
studies can be seen where some studies report a 5-fold increase in vessels compared to others. Especially in studies that did not differentiate between metastasized and nonmetastasized tumours, an expected increase by virtue of metastasized tumours would be expected to average out between studies against nonmetastasized scores, but no such trend is notable. G16 reports more microvessels overall, than G15 in metastasized tumours only. Furthermore, values for measurements in hot spots promise higher scores than measurements in any given area of the section, but L30 and H20 report more vessels overall than Hegde in hot spots21. Based on these observations, it would be impossible to establish a threshold in MVD to divide between tumours with high and low probability for metastasis, from these pooled data. Especially high densities compared to others are reported by H et al.20 possibly influenced because this is the only study to use image analysis and enhancement of staining. Those studies that evaluated metastasis, are mostly inconclusive at significance level P=0.05. Only H et al. report a significant, but counterintuitive higher density in nonmetastasized tumours20. This finding has been reported for renal carcinoma also22, but is seemingly in contradiction with the axiom by W & F. H et al. not only assessed total number of vessels, but also included diameter categories of vessels. They found that especially very small calibre
stainings were found in nonmetastasized tumours, whereas larger vessels were dominant in metastasized tumours. It is theorized that the larger vessels are functional and may contribute to metastasis, while the small ones presumably represent single endothelial cells without perfusion capability. It is not obvious if, and to what extent, regional differences in base line vascularization influence measurements31. It is conceivable that anatomic variations within the head and neck region account for some difference in MVD, but the results in the reviewed studies do not warrant a standpoint in this, because of the scattered findings. Discussion While the theory of facilitated metastasis in vessel rich tumours appears to apply for other tumours, in oral cancer debate rules. From present data, a link between high MVD and metastasis cannot be confirmed. A relation to subtypes of vessels, e.g., diameter categories, has been reported20, but this remains the only study in this field to date, and validation is desired. Identification of other subtypes, such as proliferating vessels expressing CD105, has been designated encouraging29,46. Preliminary results in our group were unfavourable, and research along that line was discontinued (H et al. unpublished data). Studies quantitating perfused vessels selectively, with Hoechst33342 or Magnetic Resonance Imaging (MRI) contrast media have not yet been applied in humans7,52.
There are many plausible theories why the assumed relation is not so straightforward in oral carcinoma, and why many studies are inconclusive. Normal oral tissue is very vessel rich, so angiogenesis may be of limited influence, when co-option suffices. Furthermore, methodological aspects introduce possible variation. Differences between immunohistochemical protocol, for instance, were found influential, but also selection of paraffin block, section within the block, and the issue of hot spot selection contributes to error. Suggested lack of reproducibility of measurements adds to variation in results. The results in this review do not allow setting of a threshold, above which metastasis is likely, and below which is unlikely, nor of intervals with low– moderate–high propensity for metastasis, because results in terms of absolute numbers lack uniformity. MVD measurements appear easy in writing, but results indicate they are not in the laboratory. It remains unknown whether all stained objects under the microscope represent vessels that contribute to metastasis, and it is uncertain whether this is important. Angiogenic activity may be a reflection of high malignant potential, not (only) because of formation of transport capable vessels, but otherwise. Hoechst33342 and MRI tumour perfusion data may help solve this issue, when compared to MVD data from the same tumour, and related to metastasis. A novel technique was reported recently, in a study using flow cytometry
The quantification of angiogenesis in relation to metastasis in oral cancer
5
microvessels can provide a means of transportation, but seemingly only predisposed cells enter, and even then, a minority establishes metastasis32,39,57. Perhaps, circulating cells complete a circle through the human body, to hatch in the lymph nodes of the neck, because of favourable soil conditions26,43. In the end, metastasis is a complex, multifactorial cascade (Fig. 2) under regulation of a number of processes, many of which play a role in other pathways. Therefore, it is unlikely that a single feature such as angiogenic potential would decide on metastasis. Angiogenesis likely facilitates metastasis, but unlikely commands it. Most data support a role for the vascular space in oral cancer metastasis, but the troublesome and precarious technique of quantitating MVD hampers its universal use as a prognosticator in clinical decision making. If flow cytometric quantification of endothelial cells3 holds up to its promise, it may well substitute MVD measurements in the near future, because of its easier handling characteristics. Fig. 2. Schematic representation of the most important steps in the metastatic cascade, and contributing interactions. ECM: extracellular matrix.
to count immunohistochemically stained single endothelial cell suspensions of colon carcinomas3. A good correlation between manual MVD and the percentage endothelial cells was observed. Although this new technique too does not allow for studying perfusion rate or differences between the number of preexistent and newly formed vessels, it is most time efficient, and carries great promise in that respect. Most studies do not address the relation blood vessels–lymphogenic metastasis. Causality between blood vessels and metastasis was adopted so wholeheartedly, that the paradox of preferential metastases in oral cancer to regional lymph nodes was overlooked. Lymph vessels are nonexistent in epithelia, and for a long time, it was assumed that there are no lymphatics in carcinomas either. Anatomical and developmental studies on lymphatics are few, and date back to the 1940s (for an overview, see ref.59). It is still not clear how lymphogenic HNSCC metastases should be viewed in the light of enhanced blood vessel supply. Antibodies to visualize blood vessels may cross react with lymphatics, or anatomical connections between blood and lymph vessels are more numerous in tumours than under normal conditions. Unique markers for
lymphatics have only recently been found4, which has hampered clear distinction between blood and lymph vessels9,27,44. One recent study comparing lymph and blood vessels in a group of HNSCCs reported a correlation between intratumoural proliferating lymphatics and lymphnode metastasis5. In 1889, Paget postulated that metastasis is not a random process, but that selected malignant cells interact at favourable sites to form daughter tumours35. Long after that report, the escape of malignant cells into the vascular compartment was regarded pivotal in meta-stasis formation. Recent findings have directed the scientific community in re-evaluating Paget’s ideas1,42. Highly sensitive detection methods have revealed that circulating malignant cells from HNSCC are much more ubiquitous than assumed until now6,62, and that shedding into the vasculature is not likely rate-limiting in metastasis. The importance of subclinical disseminated disease therefore also merits re-appraisal36. Apparently, equally important is not so much how many, but which malignant cells have disseminated13,41,50. Most recently, metastasis patent cells appeared better capable of entering into the bloodstream, than nonmetastatic tumour cells60. Tumour
Acknowledgments. The authors express their gratitude towards Prof. D. J. Ruiter for his helpful discussion.
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Address: Egied J. M. Hannen Dept of Oral and Maxillofacial Surgery Catharina-Hospital Eindhoven The Netherlands PO Box 1350 5602 ZA Eindhoven The Netherlands Tel: +31 40 2397030 Fax: +31 40 2397032 E-mail: [email protected]
Invited Review Paper Head and Neck Oncology
The quantification of angiogenesis in relation to metastasis in oral cancer: a review
E. J. M. Hannen1, D. Riediger2 1
Department of Oral and Maxillofacial Surgery, Catharina-Hospital Eindhoven, The Netherlands; 2Department of Cranio Maxillofacial Surgery, Head and Neck Surgery, University Hospital Maastricht, The Netherlands
E. J. M. Hannen, D. Riediger: The quantification of angiogenesis in relation to metastasis in oral cancer: a review. Int. J. Oral Maxillofac. Surg. 2004; 33: 2–7. 2003 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Tumours need vessels to grow into clinically significant dimensions, and to metastasize. Varying results have been reported for the relation between angiogenic activity in oral carcinomas, and the occurrence of metastasis. Quantification of microvessels in tissue sections is mostly used to assess angiogenesis, but appears subject to biases as reflected in contradicting reports. Especially the choice of immunohistochemical staining technique appears pivotal. Although microvessel density measurements (MVD) appear promising for other tumour types, in oral carcinomas no unambiguous relation could be established. Furthermore, MVD assessment appears more elaborate than expected. New, more time efficient techniques may replace MVD as we know it.
Introduction Solid tumours are dependent on vascular supply to grow beyond spheres of about 0.5–2 mm in diameter12. For this, they use existing vessels, in a process called co-option24, or they induce growth of newly sprouted vessels, i.e., angiogenesis8. Tumours with superior blood supply are thought to have a growth advantage over poorly vascularized. However, not only are bloodvessels necessary for growth, they also provide the route for possible metastasis. Here too, well-vascularized tumours are thought to metastasize more easily because of facilitated access to intravascular transportation. Ever since Folkman and Weidner postulated such56, many studies addressed this issue 0901-5027/04/010002+06 $30.00/0
in an effort to prove such a relation, and for some tumours there is a body of evidence suggestive in that direction55. But for head and neck carcinoma, varying results have been reported2,11,15,16, 17,18,20,21,23,28,30,33,34,37,38,40,47,48,49,58,61 . As a measure of angiogenic activity, most studies count the number of microvessels in tissue sections, expressed in the microvessel density (MVD)53. This technique was designated an easy prognosticator for clinical behaviour for a number of tumours54,55, but the assay proved more elaborate than expected10,14,53. In oral carcinoma too, no indisputable relation between high MVD and metastasis was found20. This paper summarizes the studies in the field of vascular quantification in relation to metastasis for oral cancer and puts
Key words: neoplasm blood supply; neovascularization; head and neck neoplasms; squamous cell carcinoma; oral carcinoma; review literature; diagnostic techniques and procedures; patient care planning. Accepted for publication 16 April 2003
results into perspective.
clinical
and
biological
Technical considerations MVD assessment was introduced by W56, and the technique basically implies counting of routine immunohistochemically stained vessel wall profiles in tissue sections of tumours25. In its original description, first at low magnification, a domain of high staining intensity in a section was identified, wherein MVD was subsequently determined at high power magnification. Theoretically, this approach introduces two biases. First, by selecting a section from the whole tumour resection specimen10, and second, by selecting a subjectively defined hot spot of angiogenic activity51.
2003 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
The quantification of angiogenesis in relation to metastasis in oral cancer Table 1. Antibodies in use for microvessel visualization in immunohistochemically stained tissue sections Antibody against CD31 (PECAM) CD34 Von Willebrand factor (F VIII-related antigen) CD105 Collagen IV Pal-E VEGF-receptors FLT-4 (VEGFR-3) LYVE-1
Epitope Cell surface antigen expressed by platelets, endothelial cells, granulocytes, some lymphocytes Transmembrane glycoprotein on vascular endothelial cells, haematopoietic precursor cells Reacts with Von Willebrand Factor in endothelial cells TGF- binding protein on endothelial cells Collagen type IV in vessel basal membrane Not known; expression exclusively on blood vessels VEGF-receptors in endothelial cells VEGF-receptor for VEGF-C, predominantly present on lymphatics Hyaluronan receptor on lymphatic endothelium
The carry of these biases apparently varies among tumour types, as some tumours prove robust enough, but for oral carcinomas there is room for doubt20. Limiting the area under investigation to be assessed, however is a practical advantage, improving time efficiency. A range of antibodies for the staining of vessel walls is available (Table 1), most of which are aimed at, but not exclusively selective for, epitopes on endothelial cells. Especially CD31, often advocated as the antibody of choice for many tumours53, is known to cross react with plasma cells, abundantly present in oral carcinomas, i.e., false positive staining may occur. Although no extensive trial comparing a broad panel of antibodies is presently available, most suited for oral specimens is CD3419,20,45. The sensitivity of staining, i.e., whether all endothelial cells are recognizably and completely stained, depends among others on the specimen fixation technique, but to a greater extent on the immunohistochemical staining procedure19. Certainly if an automated quantification technique is to be employed (vide infra), enhancement of staining is usually necessary19. Most used antibodies react both with blood vessels as well as lymph vessels (Table 1). The visual identification of the vessel transsections in the tissue section through the microscope may appear trivial if good staining quality is accomplished, but tumour vessels are mostly of aberrant morphology, tortuous, without clear lumen and with large gaps between endothelial cells. This leaves room for discussion, whether a single endothelial cell represents a true vessel wall, or not, and whether it represents a vessel suitable for transportation of malignant cells (Fig. 1). W et al.53,56 proposed to include all endothelial cells,
regardless of the above issues, and most studies comply. However, results from studies aimed at perfusion rate to quantify a tumour’s vascular dependence7,52 may deviate from MVD studies for this reason. All these considerations taken together make MVD assessment a time consuming and laborious effort. The introduction of image analysis14,19,51 improves time management, because multiple, complete tissue sections can be measured in an automated, self-
3
controlled process, reducing sampling error. Furthermore, stained objects are recognized identically in repeated measurements, eliminating intra and inter observer errors. Image analysis, however, requires excellent tissue and staining qualities: frozen sections are unsuited because of insufficient morphology. Besides, image analysis is not ubiquitously available, and requires the assistance of an image analysis expert. Overview of the literature Studies exclusively studying oral carcinoma are very few. To date, 15 studies have quantitated MVD in head and neck squamous cell carcinoma (HNSCC); some of them have studied its relation to metastasis. Despite efforts to introduce standard procedures, techniques differ among studies. Furthermore, not all studies describe all technical details satisfactorily, as to which vessels were included, and as to the definition of metastasis. Data are reported as provided in the respective studies. To facilitate comparison for this review, the results have been recalculated into number of vessels per mm2 in Table 2. Large differences between
Fig. 1. Tissue section of a tongue carcinoma, stained for CD34, with hematoxylin counterstaining19. Red profiles represent endothelial structures. Does the black arrow point at one or two vessels? Is the red stain (green arrow) a vessel, or not?
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Table 2. Results reported in HNSCC microvessel density studies, recalculated into counts per mm2 Standard Deviation (SD, where available). Values for the selected hot spot and mean overall values depending on available data
Author A2 G15 G16 G17 G17 G18 H20 H21 H21 L30 M33 M34 P37 P38 S47 T49
Tissue Oral scc HNSCC HNSCC T2–4 tongue+FOM‡ scc T2–4 tongue+FOM‡ scc T1 tongue+FOM‡ scc T1–4 tongue scc HNSCC Tongue¶ scc T1–2 tongue scc Oral scc Tongue scc Oral scc Oral scc T1 tongue scc Lip scc
Antibody F VIII CD31 CD31 F VIII CD31 CD31 CD34 F VIII or CD31 F VIII or CD31 F VIII F VIII CD31 F VIII CD31 F VIII F VIII
Mean valueSD for vessels per mm2 in hot spot Lymphnode Lymphnode+
Mean valueSD for vessels per mm2 overall Lymphnode Lymphnode+ 22.76.8
29a
71a
97.446.6 168.0 206.5 157.7 178.5 193.0 192.0 264.772.1 194.456.4 38.3 36.6
107.862.2
82.349.5
75.0/107.0*
72.9/101.0*
14271 18.3§ 14853 11624 50.013.7† 65.6/137.8*
16856 103.440.7† 59.4/118.9*
a Median; ‡floor of the mouth; *depending on magnification (200/400, respectively); †per 250 magnification field; §per 200 magnification field; ¶two cases from HN group21.
studies can be seen where some studies report a 5-fold increase in vessels compared to others. Especially in studies that did not differentiate between metastasized and nonmetastasized tumours, an expected increase by virtue of metastasized tumours would be expected to average out between studies against nonmetastasized scores, but no such trend is notable. G16 reports more microvessels overall, than G15 in metastasized tumours only. Furthermore, values for measurements in hot spots promise higher scores than measurements in any given area of the section, but L30 and H20 report more vessels overall than Hegde in hot spots21. Based on these observations, it would be impossible to establish a threshold in MVD to divide between tumours with high and low probability for metastasis, from these pooled data. Especially high densities compared to others are reported by H et al.20 possibly influenced because this is the only study to use image analysis and enhancement of staining. Those studies that evaluated metastasis, are mostly inconclusive at significance level P=0.05. Only H et al. report a significant, but counterintuitive higher density in nonmetastasized tumours20. This finding has been reported for renal carcinoma also22, but is seemingly in contradiction with the axiom by W & F. H et al. not only assessed total number of vessels, but also included diameter categories of vessels. They found that especially very small calibre
stainings were found in nonmetastasized tumours, whereas larger vessels were dominant in metastasized tumours. It is theorized that the larger vessels are functional and may contribute to metastasis, while the small ones presumably represent single endothelial cells without perfusion capability. It is not obvious if, and to what extent, regional differences in base line vascularization influence measurements31. It is conceivable that anatomic variations within the head and neck region account for some difference in MVD, but the results in the reviewed studies do not warrant a standpoint in this, because of the scattered findings. Discussion While the theory of facilitated metastasis in vessel rich tumours appears to apply for other tumours, in oral cancer debate rules. From present data, a link between high MVD and metastasis cannot be confirmed. A relation to subtypes of vessels, e.g., diameter categories, has been reported20, but this remains the only study in this field to date, and validation is desired. Identification of other subtypes, such as proliferating vessels expressing CD105, has been designated encouraging29,46. Preliminary results in our group were unfavourable, and research along that line was discontinued (H et al. unpublished data). Studies quantitating perfused vessels selectively, with Hoechst33342 or Magnetic Resonance Imaging (MRI) contrast media have not yet been applied in humans7,52.
There are many plausible theories why the assumed relation is not so straightforward in oral carcinoma, and why many studies are inconclusive. Normal oral tissue is very vessel rich, so angiogenesis may be of limited influence, when co-option suffices. Furthermore, methodological aspects introduce possible variation. Differences between immunohistochemical protocol, for instance, were found influential, but also selection of paraffin block, section within the block, and the issue of hot spot selection contributes to error. Suggested lack of reproducibility of measurements adds to variation in results. The results in this review do not allow setting of a threshold, above which metastasis is likely, and below which is unlikely, nor of intervals with low– moderate–high propensity for metastasis, because results in terms of absolute numbers lack uniformity. MVD measurements appear easy in writing, but results indicate they are not in the laboratory. It remains unknown whether all stained objects under the microscope represent vessels that contribute to metastasis, and it is uncertain whether this is important. Angiogenic activity may be a reflection of high malignant potential, not (only) because of formation of transport capable vessels, but otherwise. Hoechst33342 and MRI tumour perfusion data may help solve this issue, when compared to MVD data from the same tumour, and related to metastasis. A novel technique was reported recently, in a study using flow cytometry
The quantification of angiogenesis in relation to metastasis in oral cancer
5
microvessels can provide a means of transportation, but seemingly only predisposed cells enter, and even then, a minority establishes metastasis32,39,57. Perhaps, circulating cells complete a circle through the human body, to hatch in the lymph nodes of the neck, because of favourable soil conditions26,43. In the end, metastasis is a complex, multifactorial cascade (Fig. 2) under regulation of a number of processes, many of which play a role in other pathways. Therefore, it is unlikely that a single feature such as angiogenic potential would decide on metastasis. Angiogenesis likely facilitates metastasis, but unlikely commands it. Most data support a role for the vascular space in oral cancer metastasis, but the troublesome and precarious technique of quantitating MVD hampers its universal use as a prognosticator in clinical decision making. If flow cytometric quantification of endothelial cells3 holds up to its promise, it may well substitute MVD measurements in the near future, because of its easier handling characteristics. Fig. 2. Schematic representation of the most important steps in the metastatic cascade, and contributing interactions. ECM: extracellular matrix.
to count immunohistochemically stained single endothelial cell suspensions of colon carcinomas3. A good correlation between manual MVD and the percentage endothelial cells was observed. Although this new technique too does not allow for studying perfusion rate or differences between the number of preexistent and newly formed vessels, it is most time efficient, and carries great promise in that respect. Most studies do not address the relation blood vessels–lymphogenic metastasis. Causality between blood vessels and metastasis was adopted so wholeheartedly, that the paradox of preferential metastases in oral cancer to regional lymph nodes was overlooked. Lymph vessels are nonexistent in epithelia, and for a long time, it was assumed that there are no lymphatics in carcinomas either. Anatomical and developmental studies on lymphatics are few, and date back to the 1940s (for an overview, see ref.59). It is still not clear how lymphogenic HNSCC metastases should be viewed in the light of enhanced blood vessel supply. Antibodies to visualize blood vessels may cross react with lymphatics, or anatomical connections between blood and lymph vessels are more numerous in tumours than under normal conditions. Unique markers for
lymphatics have only recently been found4, which has hampered clear distinction between blood and lymph vessels9,27,44. One recent study comparing lymph and blood vessels in a group of HNSCCs reported a correlation between intratumoural proliferating lymphatics and lymphnode metastasis5. In 1889, Paget postulated that metastasis is not a random process, but that selected malignant cells interact at favourable sites to form daughter tumours35. Long after that report, the escape of malignant cells into the vascular compartment was regarded pivotal in meta-stasis formation. Recent findings have directed the scientific community in re-evaluating Paget’s ideas1,42. Highly sensitive detection methods have revealed that circulating malignant cells from HNSCC are much more ubiquitous than assumed until now6,62, and that shedding into the vasculature is not likely rate-limiting in metastasis. The importance of subclinical disseminated disease therefore also merits re-appraisal36. Apparently, equally important is not so much how many, but which malignant cells have disseminated13,41,50. Most recently, metastasis patent cells appeared better capable of entering into the bloodstream, than nonmetastatic tumour cells60. Tumour
Acknowledgments. The authors express their gratitude towards Prof. D. J. Ruiter for his helpful discussion.
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Address: Egied J. M. Hannen Dept of Oral and Maxillofacial Surgery Catharina-Hospital Eindhoven The Netherlands PO Box 1350 5602 ZA Eindhoven The Netherlands Tel: +31 40 2397030 Fax: +31 40 2397032 E-mail: [email protected]