Tumor Angiogenesis in Breast Cancer: Pericytes and Maturation Does Not Correlate With Lymph Node Metastasis and Molecular Subtypes

Original Study

Tumor Angiogenesis in Breast Cancer: Pericytes and Maturation Does Not Correlate With Lymph Node Metastasis and Molecular Subtypes Shreya Shrivastav,1 Amanjit Bal,1 Gurpreet Singh,2 Kusum Joshi1 Abstract The present study was undertaken to assess the structural and functional stability of tumor vasculature in breast cancer and its potential clinical relevance in reference to tumor size, tumor grade, lymph node metastasis, and molecular subtypes. Microvessel density remains the angiogenesis-related parameter associated with tumor grade and necrosis. The proliferating capillary index was the only functional parameter of angiogenesis associated with poor prognostic indicators. The microvessel pericyte coverage index (ie, pericyte coverage) did not show any correlation with the known prognostic and predictive factors. Background: Angiogenesis, traditionally assessed by microvessel density (MVD), does not give an indication of the functional status of the tumor neovasculature. The structural and functional stability of the tumor vasculature and its potential clinical relevance in breast cancer was evaluated. Materials and Methods: In invasive breast cancer, immunostaining of endothelial cells and pericytes was performed using anti-CD34 and antieplatelet-derived growth factor receptor-b antibody, respectively. Double immunostaining for the proliferating capillary index (PCI) and microvessel pericyte coverage index (MPI) was performed with CD34/Ki-67 and CD34/smooth muscle actin. Results: The mean MVD of 145 vessels/mm2 was significantly greater in grade 3 tumors (P ¼ .018) and in necrotic tumors (P ¼ .022). The PCI ranged from 0% to 17.14% (mean, 4.37%) and was associated with a high proliferative index in tumor tissue (P ¼ .044). The MPI ranged from 13.09% to 88.18% (mean, 41.35%), indicating the stability of the tumor vasculature. However, it was not significantly associated with the tumor size, tumor grade, lymph node metastasis, proliferative index, or molecular subtypes. Conclusion: MVD remains the angiogenesis-related parameter associated with tumor grade and necrosis. The PCI was the only functional parameter of angiogenesis associated with a poor prognostic indicator. The MPI did not show any correlation with the known prognostic and predictive factors. Clinical Breast Cancer, Vol. 16, No. 2, 131-8 ª 2016 Elsevier Inc. All rights reserved. Keywords: Angiogenesis, Breast cancer, Microvessel density, Microvessel pericyte coverage, Proliferating capillary index

Introduction Breast cancer is a heterogeneous disease comprising a variety of pathologic entities and a wide range of clinical behavior. The existing classification system, which depends on tumor grade, tumor stage, lymph node status, and predictive markers (eg, estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor-2 [HER2]) fails to accurately predict disease 1

Department of Histopathology Department of General Surgery Post Graduate Institute of Medical Education and Research, Chandigarh, India 2

Submitted: Apr 26, 2015; Revised: Aug 16, 2015; Accepted: Sep 11, 2015; Epub: Sep 25, 2015 Address for correspondence: Amanjit Bal, MD, DNB, Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India E-mail contact: [email protected]

1526-8209/$ - see frontmatter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clbc.2015.09.002

progression and recurrence.1,2 Two main mechanisms responsible for tumor progression are tumor angiogenesis and metastasis. In an attempt to understand angiogenesis, a prerequisite for tumor growth beyond a few cubic millimeters, most studies have used microvessel density (MVD) counting techniques to assess the tumor vasculature. The College of American Pathologists has stratified the assessment of angiogenesis by counting microvessels in immunostained tissue sections as category III, encompassing all factors not sufficiently studied to demonstrate their prognostic value.3 MVD counts assess the presence of blood vessels but do not give an indication of the functional status of the tumor neovasculature. For functional status of the newly formed tumor vessels, the vascular proliferation index with the help of Ki-67, N-methyl-2-pyrrolidone, or avb integrin and vascular differentiation and activation with markers such as LH 39 and CD105 have been used.4,5 Certain histologic surrogate markers of angiogenesis have also been studied,

Clinical Breast Cancer April 2016

- 131

Functional Status of Tumor Angiogenesis including a central fibrotic focus and the expansive growth pattern.4,5 In angiogenesis, it takes 2 to make blood vessels: endothelial cells and pericytes. Although the endothelial cells are the better characterized of the 2, pericytes are now coming into focus as important regulators of angiogenesis and blood vessel function and as potential drug targets. Activated endothelial cells sprout to form an endothelial tube, and the pericytes proliferate and encapsulate these new channels.6 These newly formed vessels that are enveloped with pericytes mature and stop remodeling.7 Pericytes, in addition to stabilizing the neovessels, are crucial for endothelial cell survival by locally releasing vascular endothelial growth factor (VEGF) and angiopoietin-1.8 The varying degrees of pericyte recruitment indicate differences in the functional status of the tumor vasculature in different tumors.9 The present study was performed to assess the structural and functional stability of the tumor vasculature in breast cancer and its potential clinical relevance in reference to tumor size, tumor grade, lymph node metastasis, and molecular subtypes.

Materials and Methods A total of 75 prospective patients undergoing lumpectomy and mastectomy during 2013 to 2014 with axillary clearance for invasive breast cancer with adequate material received were included in the

present study. In addition, 25 retrospective cases with adequate material and regular follow-up data were also included. Patients who had undergone chemotherapy before surgery and those with a history of lumpectomy followed by mastectomy for residual disease were excluded. The institute’s ethics committee approved the present study.

Histopathologic Examination All the specimens were processed for paraffin sections for routine hematoxylin and eosin (H&E) staining and immunohistochemistry. Detailed morphologic features, histologic type, grade, necrosis, lymphovascular emboli, and lymph node status were reviewed by light microscopic examination of H&E-stained sections by 2 independent observers. Special attention was given to the presence of a fibrotic focus and the growth pattern, the surrogate markers of angiogenesis (Figure 1A,B).

Immunohistochemistry for Endothelial Cells and Pericytes The details of the antibodies used are summarized in Table 1. Single Immunostaining. Endothelial cells were stained using antiCD34 antibody, and pericytes were stained using antieplateletderived growth factor receptor-b (PDGFRb). Antigen retrieval was performed using the heat retrieval method using the PT link (Dako,

Figure 1 (A) Fibrotic Focus—A Surrogate Histologic Marker of Angiogenesis (Hematoxylin and Eosin [H&E] Stain, Original Magnification 3100). (B) Infiltrating Pattern of Growth (H&E Stain, Original Magnification 3100). (C) Immunostaining for CD34, Which Stains Endothelial Cells and Identification of Vascular “Hotspots” at Low Power (CD34 Immunostain, Original Magnification 3100). (D) Microvessels Stained With CD34 Counted at 3400 (CD34 Immunostain, Original Magnification 3400)

132

-

Clinical Breast Cancer April 2016

Shreya Shrivastav et al Table 1 Antibodies and Kits Used in the Present Study Antibody CD34 SMA Ki-67 PDGFRb Secondary antibody Secondary antibody for double immunostain

Manufacturer

Clone

Dilution

Antigen Retrieval

Dako (Glostrup, Denmark) Dako (Glostrup, Denmark) Dako (Glostrup, Denmark) Cell Signaling Technology (Boston, MA) Dako Envision (Glostrup, Denmark) Dako Envision (Glostrup, Denmark)

QBEnd 10 1A4 MIB 1 28E1

1:50 1:100 1:150 1:100

Citrate (pH 6) Citrate (pH 6) Tris EDTA (pH 9) Tris EDTA (pH 9)

e

e

e

Envision G/2 Double stain System, Mouse/Rabbit (K5361)

e

e

Abbreviations: EDTA ¼ ethylenediaminetetraacetic acid; PDGFRb ¼ platelet-derived growth factor receptor b; SMA ¼ smooth muscle actin.

Glostrup, Denmark). The primary antibody was applied at room temperature for 1 hour for CD34 and overnight at 4 C for PDGFRb in a moist chamber. This was followed by a secondary antibody (Dako Envision, Glostrup, Denmark) for 40 minutes. The color reaction was developed using di-amino benzidine, and the sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted. Double Immunostaining. To study the proliferating capillary index (PCI) and microvessel pericyte coverage index (MPI), double immunostaining with CD34/Ki-67 and CD34/smooth muscle actin (SMA) was done, respectively, using the Dako Envision G/2 Double Stain System, Mouse/Rabbit (K5361). Antigen retrieval was performed by the heat retrieval method using PT link. The retrieval buffer for CD34/SMA was citrate (pH 5.9) and for CD34/Ki-67 was Tris ethylenediaminetetraacetic acid (pH 9). Additional procedures were performed in accordance with the manufacturer’s instructions.

Interpretation of Immunostaining The sections were assessed for uniformity of staining and identification of “hot spots” at low power (10), and individual microvessel counts were performed at high power (40). At least 5 independent microscopic fields per tissue section by 2 independent investigators were assessed and the following parameters evaluated: MVD, PCI, and MPI. Microvessel Density. To express the MVD counts as microscopeindependent, the counts were transformed and expressed as the number of microvessels/mm2 (1 high-powered field, 0.19 mm2). Precautions were taken to count the vessels at the center of the tumor, avoiding the periphery of the tumor, to obtain the true representation of the tumor vasculature (Figure 1C,D). Proliferating Capillary Index. To count the proliferating capillaries, Ki-67epositive endothelial cells in  5 independent highpower microscopic fields per tissue section were analyzed.10 Endothelial cell proliferation was quantified in the vascular hot spots. The PCI was determined by calculating the ratio of the number of microvessels with proliferating endothelial cells divided by the total number of microvessels (Figure 2A,B). Microvessel Pericyte Coverage Index. The MPI indicates the number of vessels that have an outer layer of pericytes and thus are

more mature vessels.10 The MPI was established by quantifying the percentage of microvessels with positive staining for pericyte markers (SMA and PDGFRb) using 2 methods: PDGFRb immunostaining and double immunostaining for CD34 and SMA. For PDGFRb immunostaining (Figure 2C,D), the density of PDGFRb-stained vessels was calculated using the same method used for MVD (ie, counting the number of vessels stained by PDGFRb in 5 high-power fields after selection of vascular hot spots and calculating its density per squared millimeter). The MPI was calculated by dividing the density of the pericytes by the MVD and multiplying that sum by 100. The second method used double immunostaining with CD34 for endothelial cells and SMA for pericytes (Figure 2E,F). The MPI was determined by calculating the percentage of microvessels that have SMA and CD34 positivity among the total number of microvessels stained by CD34. Care was taken to not count the vessels in which SMA positivity resulted from the presence of vascular smooth muscle cells. These vessels are different from pericyte-covered microvessels in that they are larger vessels with a layer of intima between the CD34-positive endothelial cells and the SMA-positive vascular smooth muscles. In the case of pericyte-covered microvessels, the endothelial cells and pericytes will be in close contact with each.

Statistical Analysis Statistical analysis was performed using the statistical package SPSS, version 17.0, for MS Windows (SPSS Inc, Chicago, IL). Correlations among the categorical variables in the multivariate and bivariate analyses were determined using the Mann-Whitney U test, Wilcoxon signed ranks test, Pearson’s c2 test, and Spearman’s rho. Significance was assumed at P < .05.

Results Clinicopathologic Parameters The clinicopathologic features of the 100 patients in the present study are summarized in Table 2. The age of the patients ranged from 30 to 74 years (mean, 49.84 years), and the age groups of 41 to 50 and 51 to 60 years included the maximum number of patients (ie, 30% and 29%, respectively). The tumor size ranged from 1 to 8 cm in the largest dimensions (mean, 2.96 cm). Most patients (65%) had a tumor size in the range of > 2 to 5 cm (stage pT2). The most frequent histologic subtype was infiltrating ductal carcinoma, no

Clinical Breast Cancer April 2016

- 133

Functional Status of Tumor Angiogenesis Figure 2 (A, B) Double Staining for Endothelial Cells by CD34 (Red) and Ki-67 (Brown) to Identify Proliferating Vessels (Double Immunostain CD34 and Ki-67, Original Magnification 3200 and 3400, Respectively). (C, D) Platelet-Derived Growth Factor Receptor b (PDGFRb)-Stained Vessels Indicating Pericyte-Covered Vessels (PDGFRb Immunostain, Original Magnification 3200 and 3400, Respectively). (E, F) Double Immunostaining for Endothelial Cells by CD34 (Brown) and Pericyte Smooth Muscle Actin (SMA) (Red). The Vessels That Stained for Both CD34 (Endothelial Cells) and SMA (Pericytes) Are Pericyte-Covered Vessels (Double Immunostain CD34 and SMA, Original Magnification 3100 and 3400, Respectively)

134

-

Clinical Breast Cancer April 2016

Shreya Shrivastav et al Table 2 Clinicopathologic Parameters Clinicopathologic Parameter

Patients (n)

Age (years)

surgery, and 50% had metastatic tumor deposits in the lymph nodes. The number of lymph nodes involved ranged from 1 to 36. Of the cases with metastatic deposits in the lymph nodes, 5 were grade 1 tumors, 29 were grade 2, and 16 were grade 3 tumors.

30-40

25

41-50

30

Molecular Subtypes

51-60

29

>60

16

The most frequently encountered subtype was luminal A (ERþ/ PRþ or PR/HER2neu), with 63 cases (63%). Of the remaining, 5% were luminal B (ERþ/PRþ or PR/HER2neuþ), 6% were HER2 overexpressing (ER/PR/HER2neuþ), and 26% were triple negative (ER/PR/HER2neu). All grade 1 tumors were luminal A. The grade 2 tumors were the most heterogeneous, with 37 luminal A, 4 luminal B, 4 HER2 overexpressing, and 8 triple negative. Most of the grade 3 tumors were triple negative (18 cases), and 14 were luminal A, 1 was luminal B, and 2 were HER2 overexpressing.

Maximum dimension pT1 (2 cm)

29

pT2 (2-5 cm)

65

pT3 (>5 cm)

6

Tumor type IDC, NST

92

ILC

4

MC

2

IMpC

1

Proliferative Tumor Index

ICC

1

The Ki-67 index ranged from a minimum of 1% to a maximum of 90% (mean, 22%). Of the 100 cases, 48 had a low proliferation index of < 14% and 52 had a high proliferation index of > 14%. The mean Ki-67 labeling index stratified by tumor grade was 13.08%, 18.58%, and 30.20% in the grade 1, 2, and 3 tumors, respectively.

Histologic surrogate markers Central fibrotic focus Necrosis >20%

35 9

Histologic grade 1

12

2

53

3

35

Lymph nodes Positive

50

Negative

50

Molecular subtypes ERþ/PRþ or PR/HER2neu

63

ERþ/PRþ or PR/HER2neuþ

5

HER2 overexpression

6

Triple negative

26

Ki-67 labeling index <14%

48

>14%

52

Abbreviations: ER ¼ estrogen receptor; HER2 ¼ human epidermal growth factor receptor-2; ICC ¼ invasive cribriform carcinoma; IDC, NST ¼ infiltrating duct carcinoma, no special type; ILC ¼ invasive lobular carcinoma; IMpC ¼ invasive micropapillary papillary carcinoma; MC ¼ mucinous carcinoma; PR ¼ progesterone receptor.

special type (IDC-NST; 92 cases). Of the remaining 6 cases, 4 were invasive lobular carcinoma, 2 were mucinous carcinoma, and 1 each were invasive micropapillary carcinoma and invasive cribriform carcinoma. Also, 35 cases had a central fibrotic focus and 9 had necrosis that involved 20% to 80% of the tumor area. Of the 92 IDC-NST cases, 51 (55.5%) were grade 2, 35 (38.0%) were grade 3, and 6 (6.5%) were grade 1 tumors. Of the 4 cases of invasive lobular carcinoma, 3 were grade 1 and 1 was grade 2. The 1 case of invasive micropapillary carcinoma was classified as a grade 2 tumor. Both cases of mucinous carcinoma and the single case of invasive cribriform carcinoma were categorized as grade 1 tumors. Axillary lymph nodes were available in all cases for examination. Of the 100 cases, 50% had no axillary lymph node metastasis at

Angiogenesis-Related Parameters The correlation of the angiogenesis-related parameters with the known prognostic parameters is presented in Table 3. Microvessel Density. The MVD of the tumors ranged from 61.05 to 368.42 vessels/mm2 (mean, 145 vessels/mm2). The mean MVD for grade 3 tumors was greater (164.05 vessels/mm2) than that for grade 2 tumors (143.55 vessels/mm2) and grade 1 tumors (109.74 vessels/mm2). This difference in MVD across the tumor grade was statistically significant (P ¼ .018). The cases with a central fibrotic focus and those with necrosis had a greater mean MVD (147.44 vessels/mm2 and 194.17 vessels/mm2) than that of those without necrosis or a fibrotic focus (138.41 vessels/mm2). This difference was statistically significant (P ¼ .022). Although the MVD was greater in the HER2 overexpressing tumors and the triple-negative breast cancer cases than in the hormone receptor-positive cases, the difference in MVD across the various molecular subtypes was not statistically significant (P ¼ .051). Proliferating Capillary Index. The PCI ranged from 0% to 17.14% (mean, 4.37%). A higher PCI corresponded with a higher proliferative index in the tumor. The mean PCI in the tumors with a high Ki-67 index was 4.62%, and the mean PCI in the tumors with a low Ki-67 index was 3.42%. This difference in PCI for tumors with a high or low tumor Ki-67 index was statistically significant (P ¼ .044). No significant association was found between the PCI and other known prognostic factors. Microvessel Pericyte Coverage Index. The MPI was evaluated using 2 methods. The PDGFR density ranged from 24.21 vessels/mm2 to 176.84 vessels/mm2 (mean, 98.14 vessels/mm2). PDGFR-stained

Clinical Breast Cancer April 2016

- 135

Functional Status of Tumor Angiogenesis Table 3 Angiogenesis-Related Parameters: MVD, PCI, and MPI in Breast Carcinoma Clinicopathologic Parameter

Patients (n)

Mean MVD (vessels/ mm2)

Size

P Value

Mean PCI (%)

.192

P Value

PDGFRb Density

.888

P Value

Mean MPI (%)

.224

.222

pT1

29

135.15

4.13

98.00

pT2

65

150

4.06

96.39

39.68

pT3

6

166.14

3.48

117.54

32.42

.018a

Grade

.139

44.17

.697

.636

1

12

109.74

5.53

97.19

42.83

2

53

143.55

3.65

100.36

41.40

3

35

164.05

Fibrotic focus Necrosis None

4.14 .022a

Morphologic feature 35

147.4

95.07 .370 105.14

39.87 40.34

194.97

2.70

100.82

138.41

4.19

93.31

.529

Positive

50

142.92

Negative

50

150

Ki-67

.326 3.75

52

146.82

4.62

97.92

Low

48

146.49

3.42

98.32

63

136.95

.259 4.39

39.68 .987

High

ERþ/PRþ or PR/HER2neu

.604 41.41

97.83 .044a

.051

41.01 .919

98.42

4.34 .978

Molecular type

.951

4.15

9

Lymph node

38.47 .152

56

.756 41.03 40.03

.859 97.19

.769 39.30

ERþ/PRþ or PR/Her2neuþ

5

123.37

4.46

96.42

39.88

HER2 overexpression

6

184.56

2.12

92.63

42.21

26

165.93

3.59

101.98

43.32

TNBC

P Value

Abbreviations: ER ¼ estrogen receptor; HER2 ¼ human epidermal growth factor receptor-2; MPI ¼ microvessel pericyte coverage index; MVD ¼ microvessel density; PCI ¼ proliferating capillary index; PDGFRb ¼ platelet-derived growth factor receptor b; PR ¼ progesterone receptor; TNBC ¼ triple-negative breast cancer. a Statistically significant difference.

microvessels often occurred in excess to the CD34-stained microvessels. No significant correlation was found between the PDGFRb density and the MVD (PCI and MPI). The PDGFRb density showed an increasing trend with tumor size and the presence of a fibrotic and necrotic focus, although this was not statistically significant (P ¼ .224 and P ¼ .152, respectively). It did not correlate significantly with the tumor grade, lymph node metastasis, or proliferative index. Triple-negative breast cancer showed greater PDGFRb density compared with the other molecular subtypes. The MPI by double staining for CD34 and SMA ranged from 13.09% to 88.18% (mean, 41.35%). The MPI calculated by double staining for CD34 and SMA was technically sounder than that calculated by staining for PDGFRb alone. Although the MPI was low in tumor with higher grades compared with that in tumors with a lower grade, the difference was not statistically significant (P ¼ .636). Also, no statistically significant difference was found between the MPI and any other known prognostic markers.

Follow-Up

136

-

Clinical follow-up data were available for 55 of the 75 prospective cases and 22 of the 25 retrospective cases. For the prospective cases, the follow-up range was 4 to 17 months (mean, 10.27 months). Among these patients, 1 developed a recurrence with cervical lymph

Clinical Breast Cancer April 2016

node metastasis at 14 months after surgery and 1 died of chemotherapy-related complications. All patients studied retrospectively for whom clinical follow-up data were available were alive and well with no recurrences or deaths. The follow-up period for this group ranged from 36 to 75 months (mean, 56.05 months).

Discussion Breast cancer is a heterogeneous disease, with numerous factors influencing its progression and outcome. To study the lessunderstood parameters that influence the behavior and outcome of breast cancer, we attempted to study tumor angiogenesis. For > 10 years, the MVD has been proposed to identify patients at a high risk of recurrence, particularly node-negative patients. Immunohistochemical staining of microvessels using a panendothelial marker followed by counting the individual vessels in “hot spots” is used to assess the MVD.11 In the present study, the mean MVD was 145 vessels/mm2, higher than that reported in other studies such as that by Kanjanapanjapol et al,12 but lower than that reported by Biesaga et al,13 in which the mean MVD was 182.9 vessels vessels/mm2. Although studies have reported significant correlations between the vessel count and tumor size in patients with breast cancer, in our study, the MVD did not increase in proportion to the tumor size.14-16 The results of the present study,

Shreya Shrivastav et al however, showed a significantly greater MVD for grade 3 tumors than for grade 2 and 1 tumors, which has not been reported by any previous studies. We also found a significantly high MVD in tumors with necrosis compared with tumors with a fibrotic focus and tumors without these 2 features. The importance of certain morphologic features such as a fibrotic focus and the growth pattern as surrogate markers of angiogenesis has been studied and associated with adverse outcomes.17 Among the molecular subtypes, although we found a greater MVD in HER2 overexpressing and triplenegative subtypes, this difference was not statistically significant. However, Mohammed et al18 found MVD to be significantly higher in triple-negative and basal-like subtypes. The functional status of tumor vasculature was evaluated by double immunostaining with CD34 and Ki-67. The mean PCI was 4.37%, which was similar to the mean PCI of 5.0% found by Eberhard et al10 but higher than that reported by Fox et al19 (2.2%) and Vartanian and Weidner et al20 (2.7%). We found a positive correlation between the PCI and a high proliferation tumor index (P ¼ .044), a finding also seen in a study by Kruger et al.21 The Ki-67 index in tumor is now an established marker of aggressive behavior of breast cancer, and a positive correlation of the PCI with the Ki67 index indicates that a high PCI is also a possible marker of aggressive behavior. We did not find any correlation between the PCI and tumor size, tumor grade, lymph node metastasis, necrosis, or fibrosis. Also, no correlation was found between PCI and the molecular subtypes in our study, although other studies have reported a positive correlation between PCI and basal-like breast carcinoma.21,22 The maturity status of the vascular bed was assessed using the MPI and double immunostaining for CD34 and SMA. We found a mean MPI of 41.35%, which was slightly lower than the mean MPI of 67.3% reported by Eberhard et al.10 The same investigators, comparing the MPI in various other tumors, found a much lower MPI of 17.9%, 12.7%, 40.8%, and 29.6% in renal cell carcinoma, glioblastoma multiforme, lung cancer, and prostate carcinoma, respectively. We did not find any significant correlation between the MPI and other known prognostic factors, including lymph node metastasis. However, Cooke et al23 reported a greater incidence of lymph node metastasis in cases with less pericyte coverage. Pericytes were also assessed by immunostaining for PDGFRb, which is considered a marker for pericytes. Various studies have shown that diminished PDGFRb expression leads to reduced coverage of vessels by pericytes.8,24,25 However, we found it to be fairly nonspecific, because it also stained the endothelial cells and stromal elements. The mean density of the vessels stained by PDGFRb was 98.14 vessels/mm2, which was much greater than the density of the SMA-stained pericyte covered vessels (39.54 vessels/ mm2). Studies have demonstrated that tumor vascular endothelial cells, a-SMAestaining cells, and peri-epithelial stroma in breast cancer might express PDGFRb.26,27 Considering these findings, it is possible that SMA-stained pericyte coverage, associated with stabilizing of the vessels, occurs only in the later stages of network formation. A more recent study using panels of pericyte markers has shown that pericytes positive for PDGFRb, VEGF receptor-1, and neural/glial antigen 2 proteoglycan and negative for a-SMA are already abundant in actively sprouting and remodeling vascular plexus; thus, their presence per se does not mark vessel stability.28

None of the currently available angiogenic markers can identify patients who are likely to respond to anti-VEGF therapies. However, some studies have shown that pericytes seem to play an important role in treatment resistance. It has been observed that even after tumor devascularization in response to VEGF inhibition, vessels heavily covered with pericytes remain. In contrast, the vessels without this “pericyte scaffold” are more susceptible to VEGF inhibition. Also, pericytes have the ability to release pro-angiogenic factors in response to PDGF; thus, the “pericyte resistance” mechanism can be overcome by the use of PDGFR inhibitors to dissociate pericytes from the endothelium.29 Pericyte coverage (ie, vessel normalization) has also been associated with improved cytotoxic drug delivery in solid tumors. This has been supported by preclinical findings that VEGF blockade facilitates deeper penetration of drugs into the tumor by restoring a hydrostatic pressure gradient across the vessel wall and inducing a more uniform distribution of blood flow.30,31 Thus, it can be hypothesized that these functional angiogenic markers can be useful in predicting the treatment response.

Conclusion On studying angiogenesis in breast cancer, we found that the MVD remains the angiogenesis-related parameter most consistently associated with aggressive behavior of the tumor in terms of tumor grade and necrosis. Tumors with a high proliferation index showed a greater PCI, and this was the only functional parameter of angiogenesis associated with a known poor prognostic indicator. Pericytes can be stained with PDGFRb and SMA, and it can be hypothesized that these stains highlight different stages of pericyte maturation. SMA-staining pericytes are associated with more mature vessels and PDGFRb staining with immature pericytes, endothelial cells, and stroma. In the present study, the angiogenesisrelated functional parameters did not show any positive correlation with the known prognostic and predictive factors and disease-free survival. However, longer patient follow-up is required to study the effect of these parameters on patient outcomes. Also, it would be worth evaluating the role of these angiogenic parameters with the response to conventional chemotherapy and targeted antiangiogenic therapy.

Clinical Practice Points  Angiogenesis is a prerequisite for tumor growth beyond a few

 

  

cubic millimeters, and most studies have used MVD counting techniques to assess the tumor vasculature. However, it takes 2 to make blood vessels: endothelial cells and pericytes. Although endothelial cells have been better characterized, pericytes are coming into focus as important regulators of angiogenesis and blood vessel function and as potential drug targets. Our study showed that the mean MVD was significantly greater in grade 3 tumors and in tumors with necrosis. The PCI was associated with a high proliferative index in tumor. The mean MPI of 41.35% indicates stabilization of the tumor vasculature; however, vasculature stabilization was not significantly associated with tumor size, tumor grade, lymph node metastasis, proliferative index, or molecular subtype.

Clinical Breast Cancer April 2016

- 137

Functional Status of Tumor Angiogenesis  We found that MVD remains the angiogenesis-related parameter

most consistently associated with aggressive tumor behavior in terms of tumor grade and necrosis.  Tumors with a high proliferation index had a higher PCI, and this was the only functional parameter of angiogenesis associated with a known poor prognostic indicator.  The MPI did not show any positive correlation with the known prognostic and predictive factors; however, vascular stabilization might play a role in chemotherapy resistance, which needs to be evaluated.

Acknowledgments The present study was supported in part by an intramural research grant from Post Graduate Institute of Medical Education and Research, Chandigarh, India.

Disclosure The authors have stated that they have no conflicts of interest.

References 1. Verma S, Bal A, Joshi K, Arora S, Singh G. Immunohistochemical characterization of molecular subtypes of invasive breast cancer: a study from North India. APMIS 2012; 120:1008-19. 2. Rakha EA, El-Sayed ME, Lee AH, et al. Prognostic significance of Nottingham histologic grade in invasive breast carcinoma. J Clin Oncol 2008; 26:3153-8. 3. Fitzgibbons PL, Page DL, Weaver D, et al. Prognostic factors in breast cancer: College of American Pathologists consensus statement 1999. Arch Pathol Lab Med 2000; 124:966-78. 4. Vermeulen PB, Gasparini G, Fox SB, et al. Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours. Eur J Cancer 2002; 38:1564-79. 5. Hasebe T, Tsuda H, Hirohashi S, et al. Fibrotic focus in invasive ductal carcinoma: an indicator of high tumor aggressiveness. Jpn J Cancer Res 1996; 87:385-94. 6. Diaz-Flores L, Gutierrez R, Varela H, Rancel N, Valladares F. Microvascular pericytes: a review of their morphological and functional characteristics. Histol Histopathol 1991; 6:269-86. 7. Fakhrejahani E, Toi M. Tumor angiogenesis: pericytes and maturation are not to be ignored. J Oncol 2012; 2012, 10 pp. 8. Abramsson A, Lindblom P, Betsholtz C. Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors. J Clin Invest 2003; 112:1142-51. 9. Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions. Circ Res 2005; 97:512-23. 10. Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res 2000; 60:1388-93.

138

-

Clinical Breast Cancer April 2016

11. Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992; 84:1875-87. 12. Kanjanapanjapol S, Wongwaisayawan S, Phuwapraisirisan S, Wilasrusmee C. Prognostic significance of microvessel density in breast cancer of Thai women. J Med Assoc Thai 2007; 90:282-90. 13. Biesaga B, Niemiec J, Ziobro M. Microvessel density and status of p53 protein as potential prognostic factors for adjuvant anthracycline chemotherapy in retrospective analysis of early breast cancer patients group. Pathol Oncol Res 2012; 18:949-60. 14. Ogawa Y, Chung YS, Nakata B, et al. Microvessel quantitation in invasive breast cancer by staining for factor VIII-related antigen. Br J Cancer 1995; 71:1297-301. 15. Bosari S, Lee AK, DeLellis RA, Wiley BD, Heatley GJ, Silverman ML. Microvessel quantitation and prognosis in invasive breast carcinoma. Hum Pathol 1992; 23: 755-61. 16. Jitsuiki Y, Hasebe T, Tsuda H, et al. Optimizing microvessel counts according to tumor zone in invasive ductal carcinoma of the breast. Mod Pathol 1999; 12:492-8. 17. Stefansson IM, Salvesen HB, Akslen LA. Vascular proliferation is important for clinical progress of endometrial cancer. Cancer Res 2006; 66:3303-9. 18. Mohammed RA, Ellis IO, Mahmmod AM, et al. Lymphatic and blood vessels in basal and triple-negative breast cancers: characteristics and prognostic significance. Mod Pathol 2011; 24:774-85. 19. Fox SB, Gatter KC, Bicknell R, et al. Relationship of endothelial cell proliferation to tumor vascularity in human breast cancer. Cancer Res 1993; 53:4161-3. 20. Vartanian RK, Weidner N. Correlation of intratumoral endothelial cell proliferation with microvessel density (tumor angiogenesis) and tumor cell proliferation in breast carcinoma. Am J Pathol 1994; 144:1188-94. 21. Kruger K, Stefansson IM, Collett K, Arnes JB, Aas T, Akslen LA. Microvessel proliferation by co-expression of endothelial nestin and Ki-67 is associated with a basallike phenotype and aggressive features in breast cancer. Breast 2013; 22:282-8. 22. Nalwoga H, Arnes JB, Stefansson IM, Wabinga H, Foulkes WD, Akslen LA. Vascular proliferation is increased in basal-like breast cancer. Breast Cancer Res Treat 2011; 130:1063-71. 23. Cooke VG, LeBleu VS, Keskin D, et al. Pericyte depletion results in hypoxiaassociated epithelial-to-mesenchymal transition and metastasis mediated by met signaling pathway. Cancer Cell 2012; 21:66-81. 24. Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 1999; 126: 3047-55. 25. Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 1997; 277:242-5. 26. Vrekoussis T, Stathopoulos EN, Kafousi M, Navrozoglou I, Zoras O. Expression of endothelial PDGF receptors alpha and beta in breast cancer: up-regulation of endothelial PDGF receptor beta. Oncol Rep 2007; 17:1115-9. 27. Bhardwaj B, Klassen J, Cossette N, et al. Localization of platelet-derived growth factor beta receptor expression in the periepithelial stroma of human breast carcinoma. Clin Cancer Res 1996; 2:773-82. 28. Witmer AN, van Blijswijk BC, van Noorden CJ, Vrensen GF, Schlingemann RO. In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor-A. J Histochem Cytochem 2004; 52:39-52. 29. Lord S, Harris A. Angiogenesis—still a worthwhile target for breast cancer therapy? Breast Cancer Res 2010; 12(suppl 4):S19. 30. Carmeliet P, Jain RK. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov 2011; 10:417-27. 31. Dickson PV, Hamner JB, Sims JL, et al. Bevacizumab-induced transient remodeling of the vasculature in neuroblastoma xenografts results in improved delivery and efficacy of systemically administered chemotherapy. Clin Cancer Res 2007; 13: 3942-50.