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Pathological evaluation of angiogenesis in human tumor
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Biomedicine & Pharmacotherapy 59 (2005) $334-$336 http://france.elsevier.com/direct/BIOPHA/
Pathological evaluation of angiogenesis in human tumor H. Sasano*, T. Suzuki Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryou-machi, 980-8575 Sendai, Japan
Abstract Angiogenesis is the process in which endothelial cells divide and migrate to form new capillaries, which support the continued growth of tumor through blood flow. Cancer-induced angiogenesis in general represents results of increased expression of angiogenic factors such as VEGF or decreased expression of anti-angiogenic factors, or a combination of both events. Numerous reported studies have demonstrated that angiogenesis plays an important role in tumor progression and metastasis of the great majority of human solid tumors. Furthermore, the quantitation of tumor angiogenesis in resected specimens of human tumor or surgical pathology specimens contribute to assessment of biological behavior and/or clinical outcome of the patients with cancer. Therefore, it is very important to assess the status of angiogenesis or cancer-induced vessels in resected tumor or surgical pathology specimens including those before and after the neoadjuvant therapy. It then becomes very important for pathologists involved in this evaluation to determine which methods to use in order to obtain accurate and reproducible results. In this short review, the status of an analysis of angiogenesis in surgical pathology specimens through analyzing vascular density or vasculatiry using immunohistochemical staining of CD34, a specific immunohistochemical marker for endothelial cells and subsequent evaluation of immunoreactivity in surgical pathology specimens will be summarized. © 2005 Elsevier SAS. All rights reserved. Keywords: Angiogenesis; Pathology; Immunohistochemistry; CD34; Vascularity
I. Introduction Angiogenesis is the process in which endothelial cells divide and migrate to form new capillaries, which support the continued growth of tumor through blood flow [1,2]. Cancer-induced angiogenesis in general represents results o f increased expression of angiogenic factors such as V E G F or decreased expression of anti-angiogenic factors, or a combination o f both events [1]. Numerous reported studies have demonstrated that angiogenesis plays an important role in tumor progression and metastasis of the great majority o f human solid tumors [3-5]. In addition, it is generally considered that selective C O X - 2 inhibitions in human tumors, both cancer and surrounding stromal cells, reduce tumor growth through suppression of new vessels formation. Furthermore, the quantitation of tumor angiogenesis in resected specimens of human tumor or surgical
* Corresponding author. E-mail address: [email protected](H. Sasano). © 2005 Elsevier SAS. All rights reserved.
pathology specimens contribute to assessment of biological behavior and/or clinical outcome o f the patients with cancer [2-4]. Therefore, it is very important to assess the status of angiogenesis or cancer-induced vessels in resected tumor or surgical pathology specimens including those before and after the neoadjuvant therapy employing selective COX-2 inhibitors. This is especially true when evaluating the possible efficacy of selective COX-2 inhibitors in neoadjuvant trials of various neoplasms including breast cancer. Not only the alterations of characteristics related to carcinoma or parenchymal cells such as the status of tumor cell proliferation or apoptosis but also that related to intratumoral vascularity as a result of COX-2 inhibition should be included in the pathological comparison between the specimens before and after the neoadjuvant therapy of the patients in order to have a better understanding of the inert response to inhibitors in individual cases. It then becomes very important for pathologists involved in this evaluation to determine which methods to use in order to obtain accurate and reproducible results.
H. Sasano, T. Suzuki/Biomedicine & Pharmacotherapy 59 (2005) $334-$336
2. CD34 immunohistochemistry With the development of highly specific immunohistochemical endothelial markers that can be easily assessed in histological archival materials, numerous quantitative studies of assessing intratumoral vascularity have been reported in various human solid tumors [3,4]. Among these immunohistochemical endothelial markers, a monoclonal antibody against CD34 has been most frequently employed in immunohistochemical evaluation of endothelial cells [3]. CD34 immunohistochemistry is generally associated with less background stain than factor VIII antigen, another frequently used immunohistochemical marker of endothelial cells and could detect single immature endothelial cells or those without lumen in the process of angiogenesis. CD34 immunohistochemistry can be also performed in automated staining instruments such as Benchmark which results in minimum inter- and intrainstitutional differences or variations of immunostaining procedures [2]. In addition, CD34 immunohistochemistry could reliably demonstrate the proliferative endothelial cells with combination of double immunostaining with MIB-1 or Ki67. Double positive endothelial cells represent proliferative endothelial cells, which may reflect the real status of angiogenesis. It then becomes very important to establish the methods of evaluating this CD34 immunoreactivity in each cases.
3. Microvascular density (MVD) Evaluation of these intratumoral vessels profiles in CD34 immunostained slides have been performed by different methods in previously reported studies of pathologic assessment in human solid tumors. The great majority of the previous studies employed microvascular density (MVD) for this purpose [1,2,4]. In this analysis, after the tissue sections were screened under the low power magnification, MVD was quantitated by counting the total number of microvessels in the most dense areas of vascularity or so-called "hot spots" in the tissue specimens [1,4]. These microvessels include not only the cells forming the lumen but also clusters of positive cells not necessarily forming the distinctive vascular lumens and single endothelial or CD34 positive cells which may be difficult to differentiate from other types of stromal cells in routine hematoxylin and eosin staining. In MVD counting, the branching vessels are usually counted as single vessels and vascularity in areas of necrosis are not evaluated. The great majority of the studies examining MVD of human solid tumors have demonstrated a statistically significant positive correlation between intratumoral MVD and clinical outcome or prognosis of the patients [1,4]. Therefore, quantitation of immunohistochemically highlighted microvessels hot spots has been considered a powerful prognostic tool of various human solid tumors. However, it is also true that in previously reported studies the antibodies used, the number and size of the hot spots evaluated, and the stratification of patients into high and low vascular groups markedly varied among these reported studies of various human solid tumors. In addition,
$335
inter- and intraobserver differences of the findings, especially selections of "hot spots of angiogenesis" in the tissue slides could represent serious drawbacks of the analysis. These features, especially subjective or non-reproducible nature of the results as well as laborious or time consuming nature of the examination itself is considered unsuitable for incorporating this method into diagnostic pathology laboratories [1]. The counting of the number of the vessels can be subjective but the evaluation of the areas of CD34 positivity can be more objective. Therefore, computer image analysis using CAS 200, which evaluate the ratio of CD34 positive areas, not the number of microvascular structures in the selected areas of the tissue sections could overcome these potential pitfalls of the analysis and could contribute to the development of much more reproducible and/or easier methods of evaluating microvascular density in surgical pathology specimens [1].
4. Chalkley method Chalkley method has been proposed as a standard method of intratumoral microvascularity and is in general considered a simple and acceptable procedure for practical evaluation of intratumoral vascularity [6-9]. In this procedure, the three or more most vascular areas or "hot spots" with the highest number of intratumoral microvessel profiles are selected subjectively from each tumor tissue sections [6-9]. A 25-point Challdey eyepiece graticule is applied to each hot-spot areas of the tumor above and oriented to permit the maximum number of points to hit on or within the areas of microvessels profiles highlighted by CD34 or other endothelial markers immunohistochemistry [6-9]. The Chalkley count represents the number of grid points that hit immunostained vessels, taken as the average from the assessment of hot spots of the tumor specimens. ChalMey method can also be combined with computerized image analysis, possibly also using the automated pixel analysis of immunohistochemical staining. This Challdey method is considered to be associated with more reproducible results than the more frequently used microvessel density method described above. In addition, results evaluated by this method demonstrated much stronger prognostic impact in a number of the tumors including invasive ductal carcinoma of the breast. Therefore, the Chalkley counting is at this juncture established a relatively rapid method of quantifying intratumoral microvascularity or cancer induced angiogenesis and may give independent prognostic information which may be useful in determining the selection of adjuvant therapy [6-9]. However, in some tumors such as prostate cancer, estimates using MVD may be more related to aggressive biological behavior of the tumor and it awaits further investigations for clarification [4]. As described above, the evaluation of intratumoral vascularity of angiogenesis in surgical pathology specimens could provide very important information. It is also very important to note that some solid human tumors were "nonangiogenic" and whatever methods employed for evaluating the vascularity of the tumor, their results are by no means related to the prognosis of the patients. It is also important to
$336
H. Sasano, T. Suzuki/Biomedicine & Pharmacotherapy 59 (2005) $334-$336
recognize that the preparation of the specimens is very important for evaluation of immunohistochemistry of endothelial markers such as CD34.
[5]
[6]
References [1]
Hasan J, Byers R, Jayson GC. Intra-tumoural microvessel density in human solid tumours. British Journal of Cancer 2002;86:1566-77.
[7]
[2]
Sasano H, Ohashi Y, Suzuki T, Nagura H. Vascularity in human adrenal cortex. Modern Pathology 1998;11:329-33.
[3]
Vieira SC, Zeferino LC, da Silva BB, Pinto GA, Vassallo J, Carasan GAF, de Moraes NG. Quantification of angiogenesis in cervical cancer: a comparison among three endothelial cell markers. Gynecologic Oncology 2004;93:12 I-4.
[8]
[4]
Rubin MA, Buyyounouski M, Bagiella E, Sharir S, Neugut A, Benson M, et al. Microvessel density in prostate cancer: lack of correlation with tumor grade, pathologic stage, and clinical outcome. Urology 1999;53:542-7.
[91
Offersen BV, Borre M, Sorensen FB, Overgaard J. Comparison of methods of microvascular staining and quantification in prostate carcinoma: relevance to prognosis. Angiogenesis in Prostate Carcinoma 2002; 110:177-85. Hansen S, Grabau DA, Sorensen FB, Bak M, Vach W, Rose C. The prognostic value of angiogenesis by Chalkley counting in a confirmatory study design on 836 breast cancer patients. Clinical Cancer Research 2000;6:139-46. Fox SB, Leek RD, Weekes MP, Whitehouse RM, Gatter KC, Harris AL. Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. Journal of Pathology 1995; 177:275-83. Offersen BV, Sorensen FB, Yilmaz M, Knoop A, Overgaard J, on behalf of the Danish Breast Cancer Cooperative Group (DBCG) Tumor Biology Committee. Chalkley estimates of angiogenesis in early breast cancer. Acta Oncologica 2002;41:695-703. Hansen S, Sorensen FB, Vach W, Grabau DA, Bak M, Rose C. Microvessel density compared with the Chalkley count in a prognostic study of angiogenesis in breast cancer patients. Histopathology 2004; 44:428-36.
8ClENOm ~DIRECT ELSEVIER
e
& BIOMEDICINE PHARMACOTHERAIS'
Biomedicine & Pharmacotherapy 59 (2005) $334-$336 http://france.elsevier.com/direct/BIOPHA/
Pathological evaluation of angiogenesis in human tumor H. Sasano*, T. Suzuki Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryou-machi, 980-8575 Sendai, Japan
Abstract Angiogenesis is the process in which endothelial cells divide and migrate to form new capillaries, which support the continued growth of tumor through blood flow. Cancer-induced angiogenesis in general represents results of increased expression of angiogenic factors such as VEGF or decreased expression of anti-angiogenic factors, or a combination of both events. Numerous reported studies have demonstrated that angiogenesis plays an important role in tumor progression and metastasis of the great majority of human solid tumors. Furthermore, the quantitation of tumor angiogenesis in resected specimens of human tumor or surgical pathology specimens contribute to assessment of biological behavior and/or clinical outcome of the patients with cancer. Therefore, it is very important to assess the status of angiogenesis or cancer-induced vessels in resected tumor or surgical pathology specimens including those before and after the neoadjuvant therapy. It then becomes very important for pathologists involved in this evaluation to determine which methods to use in order to obtain accurate and reproducible results. In this short review, the status of an analysis of angiogenesis in surgical pathology specimens through analyzing vascular density or vasculatiry using immunohistochemical staining of CD34, a specific immunohistochemical marker for endothelial cells and subsequent evaluation of immunoreactivity in surgical pathology specimens will be summarized. © 2005 Elsevier SAS. All rights reserved. Keywords: Angiogenesis; Pathology; Immunohistochemistry; CD34; Vascularity
I. Introduction Angiogenesis is the process in which endothelial cells divide and migrate to form new capillaries, which support the continued growth of tumor through blood flow [1,2]. Cancer-induced angiogenesis in general represents results o f increased expression of angiogenic factors such as V E G F or decreased expression of anti-angiogenic factors, or a combination o f both events [1]. Numerous reported studies have demonstrated that angiogenesis plays an important role in tumor progression and metastasis of the great majority o f human solid tumors [3-5]. In addition, it is generally considered that selective C O X - 2 inhibitions in human tumors, both cancer and surrounding stromal cells, reduce tumor growth through suppression of new vessels formation. Furthermore, the quantitation of tumor angiogenesis in resected specimens of human tumor or surgical
* Corresponding author. E-mail address: [email protected](H. Sasano). © 2005 Elsevier SAS. All rights reserved.
pathology specimens contribute to assessment of biological behavior and/or clinical outcome o f the patients with cancer [2-4]. Therefore, it is very important to assess the status of angiogenesis or cancer-induced vessels in resected tumor or surgical pathology specimens including those before and after the neoadjuvant therapy employing selective COX-2 inhibitors. This is especially true when evaluating the possible efficacy of selective COX-2 inhibitors in neoadjuvant trials of various neoplasms including breast cancer. Not only the alterations of characteristics related to carcinoma or parenchymal cells such as the status of tumor cell proliferation or apoptosis but also that related to intratumoral vascularity as a result of COX-2 inhibition should be included in the pathological comparison between the specimens before and after the neoadjuvant therapy of the patients in order to have a better understanding of the inert response to inhibitors in individual cases. It then becomes very important for pathologists involved in this evaluation to determine which methods to use in order to obtain accurate and reproducible results.
H. Sasano, T. Suzuki/Biomedicine & Pharmacotherapy 59 (2005) $334-$336
2. CD34 immunohistochemistry With the development of highly specific immunohistochemical endothelial markers that can be easily assessed in histological archival materials, numerous quantitative studies of assessing intratumoral vascularity have been reported in various human solid tumors [3,4]. Among these immunohistochemical endothelial markers, a monoclonal antibody against CD34 has been most frequently employed in immunohistochemical evaluation of endothelial cells [3]. CD34 immunohistochemistry is generally associated with less background stain than factor VIII antigen, another frequently used immunohistochemical marker of endothelial cells and could detect single immature endothelial cells or those without lumen in the process of angiogenesis. CD34 immunohistochemistry can be also performed in automated staining instruments such as Benchmark which results in minimum inter- and intrainstitutional differences or variations of immunostaining procedures [2]. In addition, CD34 immunohistochemistry could reliably demonstrate the proliferative endothelial cells with combination of double immunostaining with MIB-1 or Ki67. Double positive endothelial cells represent proliferative endothelial cells, which may reflect the real status of angiogenesis. It then becomes very important to establish the methods of evaluating this CD34 immunoreactivity in each cases.
3. Microvascular density (MVD) Evaluation of these intratumoral vessels profiles in CD34 immunostained slides have been performed by different methods in previously reported studies of pathologic assessment in human solid tumors. The great majority of the previous studies employed microvascular density (MVD) for this purpose [1,2,4]. In this analysis, after the tissue sections were screened under the low power magnification, MVD was quantitated by counting the total number of microvessels in the most dense areas of vascularity or so-called "hot spots" in the tissue specimens [1,4]. These microvessels include not only the cells forming the lumen but also clusters of positive cells not necessarily forming the distinctive vascular lumens and single endothelial or CD34 positive cells which may be difficult to differentiate from other types of stromal cells in routine hematoxylin and eosin staining. In MVD counting, the branching vessels are usually counted as single vessels and vascularity in areas of necrosis are not evaluated. The great majority of the studies examining MVD of human solid tumors have demonstrated a statistically significant positive correlation between intratumoral MVD and clinical outcome or prognosis of the patients [1,4]. Therefore, quantitation of immunohistochemically highlighted microvessels hot spots has been considered a powerful prognostic tool of various human solid tumors. However, it is also true that in previously reported studies the antibodies used, the number and size of the hot spots evaluated, and the stratification of patients into high and low vascular groups markedly varied among these reported studies of various human solid tumors. In addition,
$335
inter- and intraobserver differences of the findings, especially selections of "hot spots of angiogenesis" in the tissue slides could represent serious drawbacks of the analysis. These features, especially subjective or non-reproducible nature of the results as well as laborious or time consuming nature of the examination itself is considered unsuitable for incorporating this method into diagnostic pathology laboratories [1]. The counting of the number of the vessels can be subjective but the evaluation of the areas of CD34 positivity can be more objective. Therefore, computer image analysis using CAS 200, which evaluate the ratio of CD34 positive areas, not the number of microvascular structures in the selected areas of the tissue sections could overcome these potential pitfalls of the analysis and could contribute to the development of much more reproducible and/or easier methods of evaluating microvascular density in surgical pathology specimens [1].
4. Chalkley method Chalkley method has been proposed as a standard method of intratumoral microvascularity and is in general considered a simple and acceptable procedure for practical evaluation of intratumoral vascularity [6-9]. In this procedure, the three or more most vascular areas or "hot spots" with the highest number of intratumoral microvessel profiles are selected subjectively from each tumor tissue sections [6-9]. A 25-point Challdey eyepiece graticule is applied to each hot-spot areas of the tumor above and oriented to permit the maximum number of points to hit on or within the areas of microvessels profiles highlighted by CD34 or other endothelial markers immunohistochemistry [6-9]. The Chalkley count represents the number of grid points that hit immunostained vessels, taken as the average from the assessment of hot spots of the tumor specimens. ChalMey method can also be combined with computerized image analysis, possibly also using the automated pixel analysis of immunohistochemical staining. This Challdey method is considered to be associated with more reproducible results than the more frequently used microvessel density method described above. In addition, results evaluated by this method demonstrated much stronger prognostic impact in a number of the tumors including invasive ductal carcinoma of the breast. Therefore, the Chalkley counting is at this juncture established a relatively rapid method of quantifying intratumoral microvascularity or cancer induced angiogenesis and may give independent prognostic information which may be useful in determining the selection of adjuvant therapy [6-9]. However, in some tumors such as prostate cancer, estimates using MVD may be more related to aggressive biological behavior of the tumor and it awaits further investigations for clarification [4]. As described above, the evaluation of intratumoral vascularity of angiogenesis in surgical pathology specimens could provide very important information. It is also very important to note that some solid human tumors were "nonangiogenic" and whatever methods employed for evaluating the vascularity of the tumor, their results are by no means related to the prognosis of the patients. It is also important to
$336
H. Sasano, T. Suzuki/Biomedicine & Pharmacotherapy 59 (2005) $334-$336
recognize that the preparation of the specimens is very important for evaluation of immunohistochemistry of endothelial markers such as CD34.
[5]
[6]
References [1]
Hasan J, Byers R, Jayson GC. Intra-tumoural microvessel density in human solid tumours. British Journal of Cancer 2002;86:1566-77.
[7]
[2]
Sasano H, Ohashi Y, Suzuki T, Nagura H. Vascularity in human adrenal cortex. Modern Pathology 1998;11:329-33.
[3]
Vieira SC, Zeferino LC, da Silva BB, Pinto GA, Vassallo J, Carasan GAF, de Moraes NG. Quantification of angiogenesis in cervical cancer: a comparison among three endothelial cell markers. Gynecologic Oncology 2004;93:12 I-4.
[8]
[4]
Rubin MA, Buyyounouski M, Bagiella E, Sharir S, Neugut A, Benson M, et al. Microvessel density in prostate cancer: lack of correlation with tumor grade, pathologic stage, and clinical outcome. Urology 1999;53:542-7.
[91
Offersen BV, Borre M, Sorensen FB, Overgaard J. Comparison of methods of microvascular staining and quantification in prostate carcinoma: relevance to prognosis. Angiogenesis in Prostate Carcinoma 2002; 110:177-85. Hansen S, Grabau DA, Sorensen FB, Bak M, Vach W, Rose C. The prognostic value of angiogenesis by Chalkley counting in a confirmatory study design on 836 breast cancer patients. Clinical Cancer Research 2000;6:139-46. Fox SB, Leek RD, Weekes MP, Whitehouse RM, Gatter KC, Harris AL. Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. Journal of Pathology 1995; 177:275-83. Offersen BV, Sorensen FB, Yilmaz M, Knoop A, Overgaard J, on behalf of the Danish Breast Cancer Cooperative Group (DBCG) Tumor Biology Committee. Chalkley estimates of angiogenesis in early breast cancer. Acta Oncologica 2002;41:695-703. Hansen S, Sorensen FB, Vach W, Grabau DA, Bak M, Rose C. Microvessel density compared with the Chalkley count in a prognostic study of angiogenesis in breast cancer patients. Histopathology 2004; 44:428-36.