Lymphatic vessel density in radical prostatectomy specimens

Share Embed


Descrição do Produto

Human Pathology (2008) 39, 610–615

www.elsevier.com/locate/humpath

Original contribution

Lymphatic vessel density in radical prostatectomy specimens Liang Cheng MD a,b,⁎, Elena Bishop MD a , Honghong Zhou PhD c , Gregory T. MacLennan MD d , Antonio Lopez-Beltran MD e , Shaobo Zhang MD a , Sunil Badve MD a , Lee Ann Baldridge HT (ASCP) a , Rodolfo Montironi MD f a

Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA Department of Urology, Indiana University School of Medicine, Indianapolis, IN 46202, USA c Division of Biostatistics, Indiana University School of Medicine, Indianapolis, IN 46202, USA d Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA e Department of Pathology, Cordoba University, E14004 Cordoba, Spain f Institute of Pathological Anatomy and Histopathology, School of Medicine, Polytechnic University of the Marche Region (Ancona), United Hospitals, 6002 Ancona, Italy b

Received 9 February 2007; revised 7 September 2007; accepted 7 September 2007

Keywords: Prostate; Lymphatic vessel density; Biomarkers; Angiogenesis; Metastasis

Summary Formation of new lymphatic channels, or lymphangiogenesis, has been associated with poor prognosis in a number of human cancers. Its prognostic significance in prostate cancer is uncertain. We analyzed 122 radical prostatectomy specimens. Immunohistochemistry for lymphatic vessels was performed using a mouse monoclonal antibody reactive with an O-linked sialoglycoprotein found on lymphatic endothelium (clone D2-40, Signet Laboratories, Dedham, Mass). The mean lymphatic vessel densities (LVDs) of the 3 prostate compartments were compared. Lymphatic vessel densities were correlated with other clinical and pathologic characteristics. Mean values for intratumoral, peritumoral, and normal prostate LVD were 3.0, 5.2, and 4.8 lymphatic vessels per 200× field, respectively. The intratumoral LVD was significantly lower than the peritumoral or normal LVD (P b .001), and the LVD of the latter 2 compartments was not significantly different (P = .29). The prostate LVD did not correlate with other clinical and pathologic parameters. In conclusion, LVD is reduced in the intratumoral compartment compared with the peritumoral and normal prostate compartments, whereas the latter 2 have similar LVD. In contrast to other malignancies, quantitation of lymphangiogenesis in prostatic adenocarcinoma does not appear to offer useful prognostic information. © 2008 Elsevier Inc. All rights reserved.

1. Introduction

⁎ Corresponding author. Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. E-mail address: [email protected] (L. Cheng). 0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2007.09.006

The pathophysiologic mechanisms underlying lymphatic invasion and regional lymph node metastasis are incompletely understood in prostate cancer. A number of studies have documented increased expression of lymphangiogenic growth factors VEGF-C and D and their receptor VEGFR3 in prostate cancer cells, and increased expression of these

LDV density in prostate cancer genes has been linked with advanced disease stage and lymph node metastases [1-5]. Elevated levels of other angiogenic factors, such as endothelin-1, have also been found in high-grade prostatic intraepithelial neoplasia, prostatic adenocarcinoma, and in the plasma of men with prostatic adenocarcinoma [6,7]. Moreover, angiogenin expression in prostatic glands gradually increases with the onset of high-grade prostatic intraepithelial neoplasia and ultimately with the development of prostatic adenocarcinoma [7]. It seems reasonable to hypothesize that increased expression of these lymphangiogenic and angiogenic factors leads to quantitative and/or qualitative alteration of lymphatic vessels in prostate adenocarcinoma or adjacent prostate tissue in prostate adenocarcinoma–bearing prostate glands. Tumor angiogenesis is a critical component in the growth, invasion, and metastasis of solid tumors, including prostatic adenocarcinoma. Multiple studies have demonstrated increased expression of angiogenic factors, as measured by immunohistochemistry, in situ hybridization, or serum concentration, in numerous human malignancies including colon, gastric, head and neck squamous, hepatocellular, pancreatic, and urothelial carcinomas as well as melanoma, and gestational trophoblastic tumors. Moreover, in many of these cancers, prognosis has been shown to correlate with the degree of angiogenic factor expression. In prostatic adenocarcinoma, an increase in blood vessel density has been correlated with a poor prognosis. In this study, we evaluated a large cohort of radical prostatectomy specimens to investigate the degree of immunohistochemical D2-40 expression within prostatic adenocarcinoma, in peritumoral tissue, and in adjacent benign prostatic tissue. The monoclonal antibody D2-40 detects a fixation-resistant epitope on podoplanin antigen (Mv 40000 O-linked sialoglycoprotein). This protein is expressed by lymphatic endothelial cells, allowing selective identification of lymphatic vessels and assessment of lymphatic vessel density [8]. In addition, we investigated possible correlations between lymphatic vessel density in these 3 compartments and multiple clinical and pathologic parameters including age at surgery, Gleason scores, pathologic stage, tumor extent, angiolymphatic invasion, extraprostatic extension, seminal vesical invasion, lymph node metastasis, surgical margin status, presence of prostatic intraepithelial neoplasia, and perineural invasion.

2. Materials and methods 2.1. Patients Radical prostatectomy specimens (n = 122) containing invasive prostatic adenocarcinoma with adjacent normal prostate epithelium were obtained from the surgical pathology files of Indiana University Medical Center from 1990 to 1998. These cases were selected to represent the full

611 spectrum of Gleason grade and pathologic stages. The patients ranged in age from 41 to 80 years (mean, 63 years). After surgery, each prostate was weighed, measured, inked, and fixed in 10% formalin for 18 to 24 hours. After fixation, the apex and base were amputated at a thickness of 3 to 4 mm and serially sectioned at 3-mm intervals. The seminal vesicles were sectioned parallel to the junction of the prostate. The remaining prostate was serially sectioned at approximately 3- to 4-mm intervals perpendicular to the long axis of the gland from the apex of the prostate to the tip of the seminal vesicles. Standard sections, including tissue from both peripheral and transitional zones of the prostate, were prepared for histologic examination. Grading of the primary tumor from radical prostatectomy specimens was performed according to the Gleason system [9]. The Gleason scores ranged from 5 to 10. Pathologic staging was performed according to the 1997 tumor, lymph nodes, and metastasis system [10].

2.2. Immunohistochemistry Serial 5-μm-thick sections prepared from formalin-fixed, paraffin-embedded tissues from radical prostatectomy specimens were used for the study. Tissue blocks that contained the maximum amount of tumor and highest Gleason score were selected for each case, taking care to ensure that the representative blocks contained cancer of the same Gleason score as the overall score of the case, but recognizing the limitation of sample variation. Slides from these representative blocks were analyzed. Slides were deparaffinized in xylene twice for 5 minutes and rehydrated through graded ethanol solutions to distilled water. Antigen retrieval was carried out by heating sections in 0.1 mol/L citrate buffer, pH 6.0, in a pressure steamer for 20 minutes. The slides were then incubated sequentially with primary antibody (clone D2-40, prediluted antibody; Signet Laboratories, Dedham, MA), biotinylated secondary antibody, avidin-peroxidase complex, and chromogenic substrate diaminobenzidine. Endogenous peroxidase activity was inactivated by incubation in 3% H2O2 for 15 minutes. Nonspecific binding sites were blocked using Protein Block (DAKO Corp., Carpinteria, CA) for 20 minutes. Positive and negative controls were run in parallel with each batch and demonstrated that the procedure functioned properly.

2.3. Quantification of lymphatic vessel density The extent and intensity of staining were evaluated in intratumoral, peritumoral, and benign prostatic compartments from the same slide for each case. The intratumoral compartment was defined as the area encompassing all the malignant acini present in the representative hematoxylin and eosin section. The peritumoral compartment was defined as benign prostatic glands that were present in the same 100× microscopic field as the malignant glands. The area beyond the

612

L. Cheng et al.

Table 1 Correlation of intratumoral lymphatic microvessel density with clinicopathologic parameters in 122 prostate cancer patients treated by radical prostatectomy Patient characteristics

n of total No. of vessels patients (N = 122) staining w/D2-40 antibody (mean)

Age at surgery (y) b63 54 ≥63 68 Preoperative PSA (ng/mL) b4.0 46 4.0-9.9 36 10.0-19.9 46 ≥20.0 20 Prostate weight (g) b49 48 ≥49 53 Primary Gleason grade 2 2 3 61 4 34 5 25 Secondary Gleason grade 2 5 3 54 4 44 5 19 Gleason score sum b7 32 7 47 N7 43 T classification T2 59 T3a 39 T3b 24 Lymph node metastasis Positive 6 Negative 116 Extraprostatic extension Positive 60 Negative 62 Seminal vesical invasion Positive 24 Negative 98 Surgical margin Positive 65 Negative 57 Tumor extent b10% 13 10%-29.9% 45 30%-49.9% 39 ≥50% 25

P No. of vessels value staining w/D2-40 antibody (SD)

2.81 3.24

3.10

.45

3.28 2.61 3.28 3.05

3.44 2.48 3.44 2.44

.83

3.23 3.25

3.31 3.17

.90

3.50 2.61 3.68 3.24

2.12 2.75 3.32 3.65

.49

6.60 2.80 2.68 3.68

5.50 2.81 3.00 3.00

.15

3.28 2.51 3.47

3.26 2.70 3.39

.31

3.27 2.38 3.58

3.54 2.36 2.99

.27

1.17 3.15

0.75 3.15

.15

2.92 3.18

2.70 3.48

.91

3.58 2.92

2.99 3.14

.18

3.32 2.74

3.15 3.06

.27

1.85 2.80 3.92 2.76

1.77 3.21 3.26 3.03

.09

Table 1 (continued) Patient characteristics

Tumor diameter (cm) b1 1-1.9 ≥2 Lymphovascular invasion Positive Negative Perineural invasion Positive Negative High-grade prostatic intraepithelial neoplasia Positive Negative

n of total No. of vessels patients (N = 122) staining w/D2-40 antibody (mean)

P No. of vessels value staining w/D2-40 antibody (SD)

15 66 40

2.07 3.44 2.75

1.91 3.45 2.85

.41

43 79

3.56 2.77

3.19 3.05

.15

108 14

3.20 1.86

3.22 1.70

.21 .27

110 12

3.09 2.67

3.15 2.81

.66

Abbreviation: PSA, prostate-specific antigen.

peritumoral compartment was defined as the normal prostate compartment. The numbers of lymphatic vessels in these 3 areas were counted using the method described by Jaeger et al [11]. First, the slide was scanned at low power to identify the so-called hotspots: areas with the greatest density of D240-positive endothelial cells. The “hotspot region” was then scanned to identify the best fields for counting. Microscopic fields with the highest degree of immunoreactivity were chosen for analysis. Counting was performed on 3 separate 200× fields (20× objective and 10× ocular, 0.74 mm2 per 200× field) within this hotspot using an Olympus BH2 microscope (Olympus Optical, Tokyo, Japan). Any discrete D2-40positive structure, regardless of the presence or absence of a lumen, was counted as one lymphatic vessel. The microvessel density count was defined as the highest number of lymphatic vessels among three 200× fields [11].

2.4. Statistical analysis The mean lymphatic vessel densities of the 3 prostate compartments were compared using 1-way analysis of variance (ANOVA) with a random subject effect to correlate the within-subject measurements. Pairwise comparisons between the prostate compartments were made if the ANOVA revealed significant compartment effects. The intratumoral lymphatic vessel density was compared with clinical and pathologic variables using Kruskal-Wallis 1-way ANOVA. Lymph node metastasis, extraprostatic extension, tumor volume, Gleason score, and other pathologic variables were compared using Cochran-Mantel Haenszel tests for

LDV density in prostate cancer correlated ordered categorical outcomes. A P value less than .05 was considered statistically significant, and all P values were 2-sided.

3. Results Patient characteristics are illustrated in Table 1. Seventyfour percent of tumors (n = 90) had a Gleason score of 7 or higher. One hundred two patients (84%) had preoperative prostate-specific antigen elevations higher than 4 mg/dl. The final pathologic stages included T2a (9 patients), T2b (6 patients), T2c (44 patients), T3a (39 patients), and T3b (24 patients). The great majority of tumors showed perineural invasion (89%) and associated high-grade prostatic intraepithelial neoplasia (90%). Immunohistochemistry for D2-40 antibody distinguished lymphatic vessels (positive for D2-40) from endothelial-lined small blood vessels (D2-40 negative).

613 The extent and intensity of D2-40 immunostaining were evaluated in intratumoral, peritumoral, and benign prostatic compartments from the same slide for each case (Fig. 1). The highest overall lymphatic vessel densities were noted in the peritumoral compartment. The intratumoral compartment contained the fewest lymphatic channels (Figs. 1 and 2). Mean values for intratumoral, peritumoral, and normal prostate lymphatic vessel densities were 3.0, 5.2, and 4.8 lymphatic vessels per 200× field, respectively (Table 2). The intratumoral lymphatic vessel density was considerably lower than the peritumoral or normal lymphatic vessel density (P b .001), and the lymphatic vessel density of the latter 2 compartments was not significantly different (P = .29, Table 2). We also assessed whether other staging parameters showed any correlations with lymphatic microvessel density within prostatic adenocarcinoma. We found that the lymphatic vessel density did not correlate with other pathologic parameters, including lymph node metastasis,

Fig. 1 D2-40 immunostain highlights several lymphatic vessels of nonneoplastic prostate (A-B) and scarce intratumoral lymphatics (C-D). Small blood vessel in the right lower corner of image D is negative for D2-40, indicating specificity of this marker for lymphatic vessels (A). The tumor cells are also D2-40 negative (C-D). Reduced from ×100 (A, C) and ×200 (B, D).

614

Fig. 2 Lymphatic vessel density in the intratumoral, peritumoral, and normal prostatic compartments. The number of lymphatic channels is significantly reduced in the intratumoral compartment, whereas peritumoral and normal prostate compartments demonstrate roughly the same lymphatic vessel density.

Gleason score, tumor volume, extraprostatic extension, seminal vesicle invasion, or surgical margin positivity.

4. Discussion In this study, we found that lymphatic vessel density was decreased within prostatic adenocarcinoma compared with benign prostatic glandular epithelium. Moreover, lymphatic vessel density did not differ significantly between peritumoral and normal prostate compartments. Lymphatic vessel density did not correlate with Gleason score, surgical margin status, the presence or absence of extraprostatic tumor extension, seminal vesical invasion, tumor volume, lymph node status, or other prognostic parameters. Tumor angiogenesis occurs by means of a complex mechanism, which balances endothelial cell apoptosis with replication to create an increase in tumor microvascular density [12]. The cycling of endothelial cell migration, division, and differentiation results in new capillary formation [13]. The molecular elements that are most influential in angiogenesis include vascular endothelial growth factor, platelet-derived endothelial cell growth factor, basic fibroblast growth factor, thrombospondin, pleiotrophin, endostatin, and angiogenin. Angiogenesis plays a major role in the proliferation, invasion, and distant spread of malignant neoplasms, including prostatic adenocarcinoma [14,15]. Angiogenesis has been demonstrated to facilitate progression and metastasis in other tumor types, including colon, gastric, head and neck squamous, hepatocellular, pancreatic, prostatic, and urothelial carcinomas as well as melanoma, and gestational trophoblastic tumors [16,17]. Indeed, angiogenesis is felt to be requisite for tumor growth exceeding 2 mm [18].

L. Cheng et al. Whether lymphangiogenesis is as important as angiogenesis in progression of cancer is under investigation. Trojan et al [19] found that lymphatic vessel density was significantly higher in benign hyperplastic prostate tissue than in other nontumorous regions, including normal and peritumoral areas. We found that the lymphatic vessel density was significantly reduced within adenocarcinoma relative to the peritumoral and normal prostate compartments, but not significantly different between the peritumoral and normal prostate compartments. Our data suggest that prostate adenocarcinoma does not induce active lymphatic proliferation, or lymphangiogenesis, and may in fact secrete inhibitors of lymphangiogenesis. Our data seem at odds with the fact that numerous studies have revealed increased expression of several lymphangiogenic factors, including VEGF-C and D, and their receptors, in prostate adenocarcinoma cells [2-5]. On the other hand, those studies did not correlate the amplified expression of these lymphangiogenic factors with increased lymphatic vessel density in prostate adenocarcinoma. Although we did not study expression of lymphangiogenic factors in prostate adenocarcinoma, our study, together with the studies by Trojan et al [19] and Roma et al [20], provides strong evidence that active lymphangiogenesis does not play a role in lymphatic invasion and lymph node metastasis. Paucity of lymphangiogenesis is not exclusive to prostate adenocarcinoma, as analogous findings have also been reported in animal models and in other human cancers [21], most recently in breast cancer [22-24]. Lymph node metastasis has also been described in the absence of functional intratumoral lymphatics in murine melanoma and fibrosarcoma [21]. Nevertheless, it should be emphasized that restricted lymphangiogenesis in prostatic adenocarcinoma and other cancers does not contradict the potential role of these lymphangiogenic factors in lymphatic invasion and lymph node metastasis. It is quite possible that these factors may facilitate ultrastructural alterations in lymphatic channels that may make possible the entry of prostate adenocarcinoma cells into lymphatic circulation. To our knowledge, our current investigation is the first large series to examine lymphatic vessel density in intratumoral, peritumoral, and normal prostatic compartments, and to correlate the findings with multiple clinicopathologic parameters in a large series of well-characterized prostate cancer cohort. In prostatic adenocarcinoma, an Table 2 Lymphatic microvessel density within prostatic adenocarcinoma, peritumoral compartment, and benign prostatic tissue Lymphatic location

n

Mean ± SE

Range

Adenocarcinoma Peritumoral area ⁎ Benign prostate ⁎

122 122 122

3.0 ± 0.28 5.2 ± 0.29 4.8 ± 0.35

0-16 0-15 0-20

⁎ Indicates that microvessel density was higher than that within prostatic adenocarcinoma with a P value b .0001 using ANOVA.

LDV density in prostate cancer increase in blood vessel density, a measurement of angiogenesis, has been correlated with a poor prognosis. Lymphatic vessel density, on the other hand, does not appear to have any correlation with prognosis.

References [1] Gettman MT, Bergstralh EJ, Blute M, Zincke H, Bostwick DG. Prediction of patient outcome in pathologic stage T2 adenocarcinoma of the prostate: lack of significance for microvessel density analysis. Urology 1998;51:79-85. [2] Tsurusaki T, Kanda S, Sakai H, et al. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br J Cancer 1999;80:309-13. [3] Zeng Y, Opeskin K, Baldwin ME, et al. Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer. Clin Cancer Res 2004;10:5137-44. [4] Li R, Younes M, Wheeler TM, et al. Expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in human prostate. Prostate 2004; 58:193-9. [5] Jennbacken K, Vallbo C, Wang W, Damber JE. Expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 in human prostate cancer is associated with regional lymph node metastasis. Prostate 2005;65:110-6. [6] Majumder PK, Yeh JJ, George DJ, et al. Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model. Proc Natl Acad Sci U S A 2003;100:7841-6. [7] Katona TM, Neubauer BL, Iversen PW, Zhang S, Baldridge LA, Cheng L. Elevated expression of angiogenin in prostate cancer and its precursors. Clin Cancer Res 2005;11:8358-63. [8] Kahn HJ, Marks A. A new monoclonal antibody, D2-40, for detection of lymphatic invasion in primary tumors. Lab Invest 2002;82:1255-7. [9] Gleason DF, Mellinger GT. Prediction of prognosis for prostatic adenocarcinoma by combined histologic grading and clinical stage. J Urol 1974;111:58-64. [10] Fleming ID, Cooper JS, Henson DE, et al. American Joint Committee on Cancer Staging Manual. 5th ed. Philadelphia: Lippincott Raven; 1997.

615 [11] Jaeger TM, Weidner N, Chew K, et al. Tumor angiogenesis correlates with lymph node metastases in invasive bladder cancer. J Urol 1995; 154:69-71. [12] Choy M, Rafii S. Role of angiogenesis in the progression and treatment of prostate cancer. Cancer Invest 2001;19:181-91. [13] Hu G, Riordan JF, Vallee BL. Angiogenin promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities. Proc Natl Acad Sci U S A 1994;91:12096-100. [14] Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333:1751-63. [15] Izawa JI, Dinney CP. The role of angiogenesis in prostate and other urologic cancers: a review. Can Med Assoc J 2001;164:662-70. [16] Homer JJ, Greenman J, Stafford ND. Angiogenic cytokines in serum and plasma of patients with head and neck squamous cell carcinoma. Clin Otolaryngol 2000;25:570-6. [17] Ugurel S, Rappl G, Tilgen W, Reinhold U. Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival. J Clin Oncol 2001;19: 577-83. [18] Campbell S. Advances in angiogenesis research: relevance to urological oncology. J Urol 1997;158:1663-74. [19] Trojan L, Michel MS, Rensch F, Jackson DG, Alken P, Grobholz R. Lymph and blood vessel architecture in benign and malignant prostatic tissue: lack of lymphangiogenesis in prostate carcinoma assessed with novel lymphatic marker lymphatic vessel endothelial hyaluronan receptor (LYVE-1). J Urol 2004;172:103-7. [20] Roma AA, Magi-Galluzzi C, Kral MA, Jin TT, Klein EA, Zhou M. Peritumoral lymphatic invasion is associated with regional lymph node metastases in prostate adenocarcinoma. Mod Pathol 2006;19: 392-8. [21] Paera TP, Kadambi A, di Tomaso E, et al. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 2002;296: 1883-6. [22] Agarwal B, Saxena R, Morimiya A, Mehrotra S, Badve S. Lymphangiogenesis does not occur in breast cancer. Am J Surg Pathol 2005;29:1449-55. [23] Williams CS, Leek RD, Robson AM, et al. Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer. J Pathol 2003;200:195-206. [24] Vleugel MM, Bos R, van der Groep P, et al. Lack of lymphangiogenesis during breast carcinogenesis. J Clin Pathol 2004;57:741-51.

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.